Degree project

Detection of miRNAs as biomarkers to early diagnose

sepsis using human plasma from healthy donors

Bachelor Degree Project in Bioscience

First Cycle 30 credits

Spring 2023

Student: Nadin Eisa

a20nadei@student.his.se

Supervisor

Anna-Karin Pernestig

Anna-karin.pernestig@his.se

Johan Nordén

Johan.norden@his.se

Examiner: Patric Nilsson

Patric.nilsson@his.se
Abstract
One of the most common causes of death for hospitalized patients is sepsis. A host's
dysregulated reaction to infection results in sepsis, which can cause life-threatening organ
failure. Blood culturing is the current gold-standard diagnostic method for sepsis, and it might
take up to three days for the results to be verified. With the variable expression of circulatory
miRNAs observed throughout independent clinical states, miRNAs have demonstrated potential
with regard to diagnostic capacities for sepsis. As there is no longer a need for culturing, miRNA
may be measured straight from a blood plasma sample, significantly reducing the time it takes to
make a diagnosis. Two-tailed RT-qPCR, a technique that uses an RT primer with two tails, could
be the solution. The purpose of this study is to compare the effectiveness of the manual
approach with the robotic machine QIAcube for RNA extraction. Regarding the A260/A280
ratios, this study found a significant difference between human and robotic extraction of miRNA
from non-spiked plasma (p = 0.62). Additionally, it was discovered that there is a considerable
variation in the concentration of small RNA between the two approaches. In this investigation,
the two-tailed RT-qPCR technique was used to quantify miR-seps6 and test its ability to detect
and identify miRNAs in plasma samples. Using 100 μl of human blood plasma, this technique has
been established to be efficient for amplifying circulating miR-seps6. The best amplification
rates were obtained using a linear standard curve built from synthetic miR-seps 6, and the melt
curve showed a single product, which is correlated with excellent specificity. As a result of the
study's effective discovery and amplification of miR-seps 6.
List of abbreviations
cDNA - Complementary deoxyribonucleic acid
CI - Confidence interval
Cq - Quantification cycle
CRP - C-reactive protein
DNA - Deoxyribonucleic acid
HOT – Hands on time
IQR - Interquartile range
LOD- Limit of detection
NSM - Non-spiked manual extraction
NSR - Non-spiked robotic extraction
NTC – No template control
qPCR - Quantitative polymerase chain reaction
RNA - Ribonucleic acid
RT - Reverse transcription
RT-qPCR – Reverse transcription quantitative polymerase chain reaction
SD – Standard deviation
SM – Spiked manual extractions
SR – Spiked robotic extractions
TAT – Turn around time
Table of contents
Introduction............................................................................................................................................................................ 1
Sepsis.................................................................................................................................................................................... 1
Biomarkers of sepsis..................................................................................................................................................... 2
Two-tailed RT qPCR.......................................................................................................................................................3
Aim of this project................................................................................................................................................................ 4
Research questions.........................................................................................................................................................4
Materials and methods...................................................................................................................................................... 4
Research work-flow....................................................................................................................................................... 4
Preparation of plasma.................................................................................................................................................. 5
Manually extraction and elution of total RNA including miRNA...............................................................5
Robotic extraction and elution of total RNA including miRNA...................................................................6
Quality control of small RNA by using Qubit and Nanodrop.......................................................................6
Hands on time and turn around time.....................................................................................................................6
Two-tailed RT-qPCR...................................................................................................................................................... 6
Statistical Analysis..........................................................................................................................................................8
Results....................................................................................................................................................................................... 8
Quality control of the eluted RNA samples..........................................................................................................8
Comparison between the manual and the robotic method..........................................................................9
HOT and TAT for manual and robotic extractions.........................................................................................10
The two-tailed RT-qPCR method for miRNA detection and quantification........................................10
Standard curve optimization and absolute quantification........................................................................10
miRNA absolute quantitation in spiked and unspiked RNA elutes........................................................11
Melting curve analysis................................................................................................................................................14
Discussion............................................................................................................................................................................. 15
Ethical consideration....................................................................................................................................................... 19
Conclusion……………………………………………………………………………………………………………………………19
References............................................................................................................................................................................ 20
Appendix A........................................................................................................................................................................... 23
Appendix B........................................................................................................................................................................... 24
Appendix C............................................................................................................................................................................ 25
Appendix D........................................................................................................................................................................... 25
Appendix E............................................................................................................................................................................ 26
Introduction

Sepsis
In the critical care unit, sepsis is one of the most common causes of mortality for hospitalized
patients (Sakr et al., 2018). Due to some of the patients' numerous comorbidities and underlying
diseases, diagnosing is really difficult in this situation (Rello et al., 2017). Initial definitions of se
psis were defined in 1991 at a consensus meeting with an emphasis on the then-dominant
theory that sepsis was the outcome of the host's systemic inflammatory response syndrome
(SIRS) to infection. Septic shock, which was described as "sepsis-induced hypotension persisting
despite sufficient fluid resuscitation." is sepsis accompanied by organ failure and can escalate to
septic shock. Recognizing the shortcomings of these definitions. a task committee in 2001
enlarged the list of diagnostic criteria but did not propose any substitutes due to the paucity of
supporting data. In reality, for more than 20 years, the criteria of sepsis, septic shock, and organ
failure have remained substantially unaltered (Singer et al., 2016). Recent statistics show that
about 265 000 persons die from sepsis each year in the United States, where at least 1.7 million
adults are affected. More than 3.4 million instances of sepsis are thought to occur in Europe each
year, with 700 000 patients dying in hospitals and one-third of those who survive dying within a
year after being discharged (Dolmatova et al., 2020). Sepsis is still difficult to identify, detect,
and treat effectively despite the significant advancements in medical sciences and treatment
(Salomã o et al., 2019). Sepsis is characterized by end organ dysfunction, such as acute lung
injury, acute kidney injury, encephalopathy, and cardiomyopathy, as well as fever, tachycardia,
hypotension, and leucocytosis, which represent the body's reaction to infection (Dolmatova et
al., 2020). In sepsis, the immune system releases a large amount of cytokines, which can cause
damage to organs and tissues throughout the body (Delano & Ward, 2016). The immune system
plays a critical role in the development and resolution of sepsis. In the early stages of sepsis , the
immune system is activated to fight the infection. However, if the infection is not controlled, the
immune response can become excessive and lead to tissue damage and organ dysfunction
(Barichello et al., 2022). In addition. sepsis can also suppress the immune system, making the
body more susceptible to secondary infections. Researchers are actively investigating the
complex interactions between the immune system and sepsis in order to develop new
treatments and improve outcomes for patients with this condition (Hotchkiss et al., 2013). Some
potential treatments being explored include immunomodulatory therapies, which aim to
regulate the immune response to prevent it from becoming too severe or too suppressed
(National Research Council (US), 2006). The use of laboratory hematological, biochemical, and
microbiological studies is crucial in the diagnosis of sepsis (Rello et al., 2017).
Blood cultures are a crucial tool used in the diagnosis and treatment of patients who are
hospitalized. Patients who have concerns about an underlying infectious condition, whether
bacterial or fungal, frequently have blood cultures taken. Tachycardia and high fever are some of
the symptoms that may indicate that the patient has sepsis (Hotchkiss et al., 2016). Obtaining
blood cultures within an hour of admission to the hospital is advised by the most recent
surviving sepsis guidelines (Krishna et al., 2021). Identifying pathogens and choosing the best
medications depend on the results of blood cultures. (Fabre et al., 2022). Standard
microbiological blood cultures (STD) can produce erratic yields, take a long time to complete,
and have limited sensitivity, all of which can lead to the use of the incorrect antibiotics (Krishna
et al., 2021). Currently. blood culturing is used as the gold-standard for diagnosing sepsis. The de
cision on the positive or negative outcomes of an STD takes two to three days (Panday et al., 201
9). An accurate diagnosis of bacteremia or fungemia in the patient's bloodstream is made when
bacteria grow after the blood is incubated in broth. In order to control and prevent the spread of
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sepsis, it is crucial to identify the infection and the causing organism as early and properly as
possible.
Despite its low clinical positivity, a blood culture is still seen as the "gold standard" diagnostic fo
r identifying bloodstream infections (Krishna et al., 2021). A lot of work has been done recently
to identify biomarkers that will enable early identification of sepsis because culture-based
diagnosis is slow (Rello et al., 2017). For the development of quick and early diagnostic technolo
gy, early detection of the infectious agent is important (Correia et al., 2017).

Biomarkers of sepsis
A trait that is objectively tested and assessed as an indication of healthy biological processes ,
unhealthy biological processes, or pharmacological reactions to a therapeutic intervention is
described to as a biomarker. Physicians utilize biomarkers in a variety of laboratory tests to
treat and diagnose patients (Strimbu & Tavel, 2010). In clinical practice, biomarkers may also be
employed for diagnostic or prognostic uses, or in conjunction with treatment, to determine
which patients would most likely benefit from a certain treatment and to estimate its
effectiveness or harm. The usage of biomarkers is increasing, and there is a great need for
innovative molecules that can detect sepsis and septic shock (Rello et al., 2017). Researchers
have looked into six potential biomarkers. Indicative biomarkers for sepsis include PCT and CRP.
One of the first biomarkers was the acute inflammatory phase protein known as C-reactive
protein (CRP), which is produced by the liver. Tillet and Francis first identified CRP in 1930.
Procalcitonin (PCT), which was first identified in the 1970s, is a precursor to calcitonin
generated by the C-cells of the thyroid gland and is linked to life-threatening bacterial infections
(Hung et al., 2020).
Endogenous miRNAs are a class of small non-coding RNA molecules that are involved in the
post-transcriptional control of gene expression and RNA silencing. Endogenous miRNAs are
transcribed from DNA and undergo a series of processing steps that result in the production of a
mature miRNA, which can then bind to target mRNAs and regulate their expression (Dexheimer
& Cochella, 2020). Endogenous miRNAs play important roles in a wide range of biological
processes, including development, cell differentiation, and disease. They are found in virtually all
eukaryotic organisms (Annese et al., 2020). Most miRNAs are found within cells, some have been
detected in various bio-fluids including blood plasma and serum, urine, and cerebrospinal fluid
(Condrat et al., 2020). These extracellular miRNAs are known as circulating miRNAs and they
have shown stable expression levels in bio-fluids, even under harsh conditions like high pH and
boiling (Sohel, 2016). Circulating miRNAs have become an attractive candidate as potential
biomarkers for various diseases due to their stability in bio-fluids (Kumar & Reddy, 2016).
However, cellular miRNAs tend to degrade rapidly in bio-fluids, particularly in plasma, which is
likely due to the presence of RNases (Condrat et al., 2020).
Several mechanisms have been proposed to protect circulating miRNAs from RNase activity,
including encapsulation within exosomes, integration into AGO ribonucleoprotein complexes, or
a combination of both (Ortiz-Quintero, 2020). Exosomes are small membrane-bound vesicles
that are involved in intercellular communication, and they are thought to protect miRNAs by
shielding them from RNase activity. AGO proteins on the other hand are part of a
ribonucleoprotein complex that is involved in miRNA-mediated gene silencing (Théry, 2011).
When miRNAs are incorporated into AGO complexes, they become stabilized and less
susceptible to degradation (Park et al., 2017). The stable expression levels and resistance to
degradation of circulating miRNAs make them a promising biomarker for several diseases,
including sepsis (Benz et al., 2016). Researchers have investigated the potential of specific
circulating miRNAs as biomarkers for sepsis diagnosis, prognosis, and monitoring treatment

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response. Researchers are looking at miRNAs and ncRNAs as sepsis biomarkers (Barichello et
al., 2022). Numerous studies have been done on how miRNAs regulate inflammatory reactions.
Microarray screens were used to examine the expression of serum miR-574-5p in 12 surviving
and 12 non-surviving sepsis patients in order to assess the clinical prognostic predictive value of
these dynamic blood miRNAs for sepsis patients. The findings demonstrated that miR-574-5p
was superior to any single indication in prognostic predictions for sepsis patients' mortality.
Numerous of these studies give credence to the idea that miRNAs can decrease the inflammatory
response (Huang et al., 2019). MiRNAs can be challenging to extract and quantify and detect
from biofluids like plasma. However, recent research has shown that miRNA can be quantified
using the Qubit® microRNA assay and extracted from plasma samples using both the manual
extract and QIAcube robotic machine, miRNA was also detected and quantified using the two-
tailed RT-qPCR (Von, 2020).

Two-tailed RT qPCR
To study miRNA expression levels, researchers use a process called reverse transcription (RT) to
convert miRNA molecules into complementary DNA (cDNA) molecules that can be amplified and
detected using quantitative polymerase chain reaction (qPCR) (Zhang et al., 2019). Two main me
thods are commonly used for miRNA RT: stem-loop RT and two-tailed RT. In stem-loop RT, a ste
m-loop primer is designed to anneal to the miRNA sequence of interest, which is then reverse tra
nscribed into cDNA using a reverse transcriptase enzyme (He et al., 2021). The resulting cDNA c
an then be amplified and quantified using qPCR. In contrast, two-tailed RT uses a specially desig
ned oligonucleotide primer that has a hairpin loop structure with two hemiprobes that are comp
lementary to different sections of the target miRNA. This approach has been shown to have impr
oved specificity and sensitivity compared to stem-loop RT (Androvic et al., 2017). The fact of cD
NA can be amplified and quantified using qPCR, did allow researchers to measure miRNA expres
sion levels in various tissues or biofluids, such as blood, urine, or saliva. In the case of sepsis, res
earchers have identified several circulating miRNAs that show differential expression levels in s
eptic patients compared to healthy individuals, suggesting their potential as diagnostic or progn
ostic biomarkers for sepsis (Precazzini et al., 2021).
The three most popular methods for determining miRNA expression are microarrays, next-
generation sequencing (RNA-Seq), and reverse transcription quantitative PCR (RT-qPCR). RT-qP
CR is a procedure that is usually utilized when high levels of accuracy and precision are require
d, in addition to the confirmation of data from screening tests. It is also the preferred strategy w
hen just a small number of objectives are quantified, especially when the available material is li
mited (Androvic et al., 2017). The uncomplicated process is another attractive element that is si
mple to set up in labs with RT-qPCR experience. With a sensitivity good enough to identify as litt
le as ten target miRNA molecules, two-tailed RT-qPCR has a dynamic range of seven logs. So far,
a number of RT-qPCR-based techniques for miRNA quantification have been created , several of
which are commercially accessible (Androvic et al., 2017). In general, the techniques fall into tw
o categories: universal reverse transcription and specific reverse transcription. The two-tailed R
T primers are made up of an oligonucleotide tether that has been folded into a hairpin and two h
emiprobes that are complementary to different sections of the target miRNA (Voss et al., 2021).
With this innovative design, the RT primer's binding to its template is strengthened, increasing s
ensitivity. The 3′-hemiprobe can be brief, giving mismatches in the 3′-region significant discrimi
nating power, and allowing for the construction of miRNA-specific qPCR primers with a high eno
ugh melting temperature (Tm). When the divergent nucleotide is present in the middle or near t
o the 5′ end of the miRNA sequence, the 5′-hemiprobe enhances the ability to distinguish betwee
n extremely similar sequences (Androvic et al., 2017).
Total RNA extraction was carried out in this experiment using two alternative techniques:
Page: 3
manually and robotic using a QIAcube (Qiagen) and the miRNeasy® Serum/Plasma Advanced
kit protocol (2021). This kit is being used to determine whether the extraction method will
produce miRNAs of higher quality and quantity to use as biomarkers for sepsis diagnosis.
In clinical laboratory diagnostic work, the extraction technique with greater assessment might
be employed as a guideline. Studying the quantity and quality values of small RNA is important
to determine if they have an impact on the two-tailed RT-qPCR result. The manual extraction
technique is still an option to utilize if it is doing well enough. However, the clinical lab is much
more interested in having robots there. In this thesis project, the robotic machine QIAcube will
be utilized in order to evaluate the outcomes of both methods the robotic extraction and the
manual extraction. This will investigate if the robotic method may reduce the amount of sample
volume required for diagnosis, save time, and effort, and reduce the risk of cross-contamination.
This work intends to assess the quality of the small RNA by the two extraction strategies and
optimize the two-tailed RT-qPCR technology in order to identify and quantify miRNAs extracted
from human plasma. This detection is promising to be used as biomarkers in a future multi-
marker panel for the early identification of sepsis.

Aim of this project

Dr. Anna-Karin Pernestig. Senior Lecturer in Biosciences at the University of Skö vde in Sweden,
is the lead researcher on several active studies in the study of sepsis, including this one. In order
to develop a diagnostic kit for sepsis, the aim of this thesis research was to examine the usage of
miRNA in a panel of biomarkers for earlier detection of sepsis. In order to diagnose patients with
bacterial sepsis earlier than is typically the case today. Pernestig and her coworkers are focusing
on proteins, microRNA, and the vital parameters as biomarkers. Consequently, focusing on these
vital parameters raises the possibility of the patient surviving sepsis and less comorbidities.
This thesis project is not the only one currently ongoing at the University of Skö vde; earlier
initiatives there have shown the feasibility of utilizing miRNA as a biomarker to identify sepsis in
its early stages. MiRNA candidate miR-Seps 6 will be spiked into plasma and extracted using two
methods: manually and robotic. By comparing manual and robotic extraction techniques, the opt
imal strategy to extract these candidate for two-tailed RT-qPCR will be discovered. The develop
ment of early and quick diagnostic technology depends on the improvement of the method for th
e extraction of the total RNA; early detection of the infection that causes sepsis is essential to ass
ist healthcare personnel in treating people correctly. Consequently, this may lead to patients rec
eiving the proper medication before it spread in the patient's body. This will also help to easily d
iagnose if the patient has a bacterial infection that needs to be treated with an antibiotic.

Research questions
This project will attempt to address a number of scientific research questions. including the
following:
 Is it more effective to extract total RNA including small RNA from human plasma using a
robotic machine or a manual method?
 Is there a similar yield and quality of total RNA including small RNA using the robotic
machine compared to manual extraction techniques?
 Is it possible to detect and quantify the extracted total RNA including small RNA from
human plasma samples using the two-tailed RT-qPCR technique? Is there any quantification
difference between the two extraction methods; manual and robotic methods?

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Materials and methods
Research work-flow
Total RNA extraction and miRNA detection were the main goals of this research. The research
focused on the naturally occurring miRNA in plasma from healthy adult donors as well as plasma
from healthy donors that had been spiked with candidate miR-seps 6 (integrated DNA
Technologies). 32 samples total were used for the complete thesis project. The Appendix section
contains a listing of all samples by name and type. The project's process is described in (Figure
1).

Figure 1. the whole thesis project's procedures. The number of samples from each of the three groups is
shown in the picture. Overall (n=32). Both extraction techniques followed the miRNeasy® Serum/Plasma
Advanced kit protocol from Qiagen. Each sample used to extract the RNA eluate was around 15 μl. MiRNAs
were detected using two-tailed RT-qPCR technique. All the identified miRNAs were absolute quantified
after data processing.

Preparation of plasma
Human Plasma samples from healthy donors have been stored at the University of Skö vde in a -8
0 °C freezer. The protocol miRNeasy Serum/ Plasma Advanced Kit Handbook 2021 recommenda
tion was to incubate at 37 degrees in a water bath until samples are completely solved and salts
are dissolved. The plasma samples have been thawed on ice and later incubated in 37°C water ba
th VWB 2 (VWR) for 20 seconds.

Manually extraction and elution of total RNA including miRNA

For the purification of the total RNA including miRNA, miRNeasy®Serum/Plasma Advanced kit
Protocol 2021: Purification of Total RNA, including miRNA, from Serum and Plasma (Qiagen) wa
s followed with some changes. According to the mentioned protocol, the starting amount of plas
ma is 200 μl. Despite this, 100 μl of plasma was used in this project for each sample. This adjust
ment resulted in a decrease in RPL and RPP buffers. It was decided to use 30 μl of RPL buffer and
10 μl of RPP buffer instead of 60 μl of RPL and 20 μl of RPP, respectively. The serial dilution 1:10
fold was performed to spike-in the spiked extractions with the candidate miR-Seps 6 (Integrated
DNA technologies). Candidate miR-Seps 6 was diluted with RNAse-free water (VWR life science)
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to get the copy numbers 10^7 copies/μl, 10^6 copies/μl and 10^5 copies/μl. Candidate miR-Sep
s 6 original stock has the copy number 10^13 copies/μl. This stage was finished prior to beginni
ng the extraction procedure. In advance of adding the RPP buffer to the samples, the spike-in ste
p was performed. With the exception of the spike in, step number four in the protocol miRNeasy
®Serum/Plasma Advanced kit Protocol 2021: Purification of Total RNA, including miRNA, from
Serum and Plasma (Qiagen), non-spiked plasma samples were extracted using the same method
s as spiked-in plasma samples. For both spiked in samples and non spiked samples 100 μl of Isop
ropanol was added and the RNA was eluted with 15 μl of RNAse-free water for all samples durin
g the process. These preformed manually.

Robotic extraction and elution of total RNA including miRNA

For RNA extractions using the robotic method, with and without spikes total RNA including miR
NA was extracted robotically using QIAcube (Qiagen). Steps one until six in the protocol miRNea
sy®Serum/Plasma Advanced kit Protocol 2021: Purification of Total RNA, including miRNA, fro
m Serum and Plasma (Qiagen) was completed manually. Steps seven through thirteen were carri
ed out by the QIAcube (Qiagen) equipment, which also was set up to elute the RNA with 15 μl of
RNAse-free water. A further procedure has been included in step 1 which is adding 100 μl of wat
er to 100 μl of plasma.

Quality control of small RNA by using Qubit and Nanodrop

The quantity and purity of all spiked and unspiked eluates were evaluated after the extraction te
chnique. 1 μl of each eluate was used to test the purity of the total RNA extraction using a Ds-11
spectrophotometer (Denovix). All of the samples were evaluated in triplicate for the purity analy
sis. The Qubit®microRNA assay kit (Thermo Fisher) was used to measure the quantity of small
RNA using the Qubit 4.0 Fluorometer (Thermo Fisher). Additionally, the Qubit 4.0 fluorometer
(Thermo Fisher) was utilized to conduct triplicate measurements using 1 μl of each eluate. The e
luates were kept at -20°C following the final step of the quantity and purity examinations.

Hands on time and turn around time

The time required to prepare the two RNA extraction procedures has been calculated using two
different ways. The first method, hands-on time, counted time spent performing manual and
handwork. The fixing time noted in the procedure is not included in the hands-on time
technique. The incubation and centrifuging processes are involved in this fixing time. The turn-
around time approach was also measured by figuring out how long it took to complete the entire
extraction from beginning to end. The time was calculated using a digital watch and the time was
taken during the extraction of five non-spiked plasma samples.

Two-tailed RT-qPCR
The two-tailed RT-qPCR was used to detect and quantify the miRNA in the spiked and un-spiked
samples. However, additional procedures have to be followed in order to perform the two-tailed
RT-qPCR detection. CDNA synthesis and reverse transcription (RT) of miRNAs were carried out.
The two-tailed RT-reaction was prepared for the candidate miR-Seps 6 (Integrated DNA technol
ogies) and for the final extracted samples, these were reverse transcribed into cDNA templates u
sing the GrandScript cDNA FreePrime kit (TATAA Biocenter) and using the protocol for two-taile
d priming. However some changes were adjusted to this protocol and followed as mentioned in
(Table 1) and (Table 2).

Absolute quantification
The two-tailed RT-reaction was prepared for the candidate miR-Seps 6 (Integrated DNA technol

Page: 6
ogies) to preform a standard curve according to (Table 2). The cDNA of the candidate miR-Seps
6 which has 10^12 copies/μl, were serially diluted in nuclease-free water at a 1:10 fold ratio. Th
is was preformed to obtain a standard curve. Eleven samples with 10^11 copies/ μl to 10^2 copi
es/μl miRNA copies were produced by diluting the cDNA. A standard curve was created by using
the serial dilution.
Table 1. RT-reaction.
Component Volume (μl)
10 μl/reaction
Grandscript freeprimer mix 5x 2
(TATAA Biocenter)
GPS Enhancer 10x 1
GrandScript RT enzyme 0.5
RT primer (0.2 μM) 2.5
miRNA sample 4

Table 2. RT-reaction for standard curve applied for absolute quantification.

Component Volume (μl)
10 μl/reaction
GrandScript FreePrime Reaction 2
Mix (5x)
GPS Enhancer 10x 1
GrandScript RT enzyme 0.5
RT primer (0.2 μM) 2.5
Nuclease-free water 3
miRNA (10^13 copies/μl) 1

The two tailed-RT reactions were run in the PCR machine (Biometra). According to the TATAA G
randScript cDNA FreePrime kit (TATAA Biocenter), the program that has been utilized in the PC
R machine involves 45 minutes at 42 °C, followed by 5 minutes at 85 °C and cooled at 4 °C. These
procedures were followed each time qPCR had to be performed to detect and quantify miRNA. Af
ter the cDNA samples were prepared, they were kept at -20 °C to be used later in making the ma
ster mix for qPCR.
Using the two-tailed RT-qPCR protocol and TATAA SYBR® GrandMaster® Mix (TATAA Biocente
r), the generated cDNA templates from the two-tailed RT reactions of standard curve preparatio
n, spiked and unspiked RNA eluates were quantified. Preperation of the master mix was followe
d as mentioned in (Table 3).
Table 3. The master mix components for qPCR using SYBR grandmaster mix kit.
Component Volume (μl)
8 μl/reaction
TATAA SYBR® GrandMaster® 5
Mix (2x)
Forward primer (10 μM) 0.2
Reverse primer (10 μM) 0.2
Nuclease-free water 2.4
ROX dye (500 μM) 0.2

According to (Table 4), several cDNA preparations were carried out. Instead of using 2 μl of cDN
A template and 8 μl of master mix to make NTC as control test to check the contamination. 10 μl
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of the master mix was added in total. Additionally, each sample was placed as triplicates. The 96-
well plates (Applied Biosystems) were vortexed by an Analog vortex mixer (VWR) and spun dow
n using a plate centrifuge (Nippon Genetics Europe) for 20 seconds before running the qPCR rea
ctions. Following that, plates were placed in the Ariamx Real-Time PCR system (Agilent Technol
ogies) qPCR machines.
Table 4. The amount of the needed components to run the qPCR machine.
Component Standard curve* Eluted samples*
Volume (μl)
Mastermix 31.5 28
cDNA 3.5 7
* Mentioned volumes (μl) are enough for 3.5 reactions.
After the qPCR was completed, absolute quantification was then performed by comparing the m
ean Cq value of the technical triplicates from the tested samples to the standard curve. This was
done to determine the miRNAs' unknown quantities. The linear standard curve created from mi
Rseps-6 cDNA was utilized to accurately measure the circulating miR-seps 6 contained in the elu
−1
ates of non-spiked samples. It was calculated using the formula y=mx+b . The formula E=
m
was used to calculate the linear standard curve's efficiency. And % E=(E−1)×100 was used to
calculate the percent efficiency. The log concentration were determined by using the formula
( y−41.88)
( )
(−3.243) . The log concentration obtained by adding the Cq-values as y in the mentioned
x=10
equation above. X= The log concentration.

Statistical Analysis
All statistical analysis was performed using IBM SPSS 27 for Windows. The significance threshol
d was set at (p=0.05). The Shapiro-Wilk test was employed in all statistical studies to confirm no
rmality, and the appropriate statistical test was then used based on the hypothesis being tested.
Since the data were not normally distributed, the Mann-Whitney U test was utilized to compare
robotic and manual extractions concentration and purification. In order to determine where the
extractions would be placed on the linear standard curve and to determine which log copy
number the extractions have, calculations relating to the linear standard curve were performed
using Microsoft Excel for Microsoft 365. Also, by using Excel, the mean was calculated for all the
data mentioned in the Appendix.

Results
Quality control of the eluted RNA samples
When the extraction process for both spiked and non-spiked plasma samples was done manually
and using QIAcube, the purity and the quality of the extractions have been measured. The raw
data of all samples are mentioned in (Appendix A,B,C,D and E). To check the result for the
concentration of the small RNA and the purity ratio of the total RNA extracted, the mean and the
standard deviation were measured (Table 5).
Table 5. Mean values and standard deviation of the quality and purity of small RNA for both
spiked and non-spiked extracted samples.
Sample Purity of the total RNA ratio Small RNA concentration
A260/280 Mean (ng/μl) ± SD
Mean ± SD
SM10^5 (n=5) 1.29±0.24 0.58±0.19*

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 SM10^6 (n=1) 1.27±0.30 0.79±0.06
SM10^7 (n=1) 1.26±0.28 1.41±0.12
SR10^5 (n=5) 1.42±0.45 0.90±0.38*
SR10^6 (n=1) 1.58±0.12 0.50±0*
SR10^7 (n=1) 1.66±0.08 0.50±0*
NSM (n=9) 1.32±0.53 1.80±1.33
NSR (n=9) 1.46±0.15 4.88±0.22
* Out of range samples were set to 0.50 ng/ μl according to the detection range in Qubit. The results of the
mean±SD of small RNA concentrations and total RNA purity absorbance ratio 260/280 for spiked and
non-spiked RNA eluates manually and robotically are mentioned above. Detailed results are in the
appendix.

Comparison between the manual and the robotic method

To compare the result of the two methods extractions, manually and robotic extraction, both
quantity of small RNA and purity analysis was done.
The median concentration for the non-spiked manual samples (NSM) (n=9) was 0.78 ng/μl
(IQR=2.44), and the median concentration for the non-spiked robotic samples (NSR) (n=9) was
4.94 ng/μl (IQR=0.36). To test the normality distribution for both the robotic and the manual
extractions, a Shapiro-Wilk test was run. The test has shown that the robotic extractions have a
significance level (p=0.046), and the manual extractions have a significance level (p=0.001)
which means they are not normally distributed. The results should be normal distributed if the
p-value of the data is larger than 0.05. A box plot that proceeded showed a significant distinction
between the distribution of concentrations between the NSM and NSR (Figure 2A). Also, the
Mann-Whitney U test was chosen to compare the two variables between the NSM and NSR in
order to determine whether there was a significant difference in the concentration of small RNA
obtained from each technique. The p-value is 0.001, which means that there is a significant
difference between NSR and NSM samples for the distribution of the small RNA concentration.
The same procedures used to compare the concentrations were also used for the A260/A280
ratio for both the NSM (n=9) and NSR (n=9). This was done to determine whether there is any
difference in the purity between the NSR and NSM. The median A260/A280 ratio for NSM
samples (n=9) was 1.21 (IQR=0.90), compared to the median A260/A280 ratio for NSR samples
(n=9) was 1.48 (IQR=0.15). NSR and NSM samples differ significantly from one another. As a
result of the Shapiro-Wilk test, the robotic extractions have a significance level (p=0.012), and
the manual extractions have a significance level (p=0.05). In order to determine if there was a
significant difference in the total RNA purity obtained from the two procedures, the Mann-
Whitney U test was chosen to compare the two variables. NSM and NSR. As a result, when
comparing the distributions of the A260/A280 ratios between the NSM and NSR samples, the
Mann-Whitney U test indicated statistically significant difference (p=0.62). However, there is
one outlier marked with (⚬) and another extreme outlier marked with (*) (Figure 2B).

A)

Page: 9
B)

Figure 2: The interquartile range (IQR) is displayed in each box. Each box displays the median of the data
as a horizontal black line. A 95% confidence interval can be seen on the vertical line. A) Concentrations of
small RNA extracted manually (NSM) (n=9) and via the robotic method QIAcube (NSR) (n=9). B) RNA
purity absorbance ratio 260/280 extracted manually and via the robotic method QIAcube.

HOT and TAT for manual and robotic extractions

Five non-spiked plasma samples were used to determine the hand-on time and turn-around
time for both extraction techniques (Table 6). The hand-on time represent the total amount of
time required to complete the extractions for the five samples without any waiting time.
Incubation time and centrifuging time are exampled of the waiting time. However, the turn-
around time is the period of time it takes from the start point of the extractions to the end point.
The end point of these two ways of time measurement is the final phase of the extraction
procedure.
Table 6. Required hands-on time and turnaround time for manual and QIAcube RNA extraction
processes.
Method Hand-on time Turn-around time
(minutes) (minutes)
(n=5) (n=5)
Manually 44 67
QIAcube 17 34

Page: 10
The two-tailed RT-qPCR method for miRNA detection and quantification

Standard curve optimization and absolute quantification

Absolute quantification was utilized to estimate the copy number of miRNA present in the
plasma samples, to preform this a standard curve was required. To obtain the standard curve ,
the mean of the Cq values of triplicate with different miRseps-6 amplification copy numbers
were plotted on the y-axis and the log, concentration of the miRseps-6 with various copy
numbers on the x-axis to create a standard curve (Figure 4A). The data for both before and after
outlier removal were evaluated together with the indicated qPCR optimization parameters
(Table 7). Two outliers have been removed to provide an acceptable standard curve (Figure 4B).
A)

B)

Figure 4: A) the standard curve before deleting the 10^3 and 10^2 copies of miR-Seps 6. B)
shows the standard curve after removing the two outliers.
Table 7. shows the values for the different criteria before and after removing the outliers 10^3
and 10^2.
Criteria Before outlier removal After outlier removal
Coefficient of 0.94 0.99
determination (R^2)

Page: 11
 Efficiency precentage 142.9% 103.3%
(%)

Slope (m) -2.59 -3.25

miRNA absolute quantitation in spiked and unspiked RNA elutes

With the intention of ensuring the effectiveness of the quantitative amplification by two-tailed
RT-qPCR (TATAA Biocenter), the plasma samples were spiked with candidate miR-Seps 6 were
manually extracted SM (n=3) and three additional eluates with various copy numbers were
robotic extracted using QIAcube SR (n=3). The copy numbers were 10^7 copies/μl, 10^6
copies/μl and 10^5 copies/μl. To ensure the effectiveness of the quantitative amplification by
two-tailed RT-qPCR (TATAA Biocenter), these RNA eluates were employed as a positive control
(Table 8A). Results from manual NSM (n = 9) and QIAcube NSR (n = 9) extractions were
compared among 18 Non-spiked RNA eluates in total (Table 8B).
The standard curve in (Figure 5) has the outcome of the log concentration and the Cq-value of all
samples mentioned in (Table 8A and 8B) added to it. This was done to determine where will
these samples be placed. As result, some of the certain non-spiked samples that were manually
extracted are considered outliers, which means that they are not plotted on the standard curve
with other data.
Table 8A. Absolute quantification of candidate miR-Seps 6 in spiked RNA eluates.*
Spiked samples Mean Cq ± SD Log concentration
With 10^5 copies/μl
SM1 26.19±0.02 6.89E+04
SM2 27.81±0.14 2.18E+04
SM3 26.66±0.11 4.93E+04
SM4 27.97±0.12 1.95E+04
SR1 28.06±0.28 1.83E+04
SR2 27.77±0.26 2.24E+04
SR3 28.06±0.16 1.83E+04
SR4 27.96±0.16 1.96E+04
* In this table, samples marked as SR refer to spiked plasma samples that were extracted using a robotic
technique. The SM are manually extracted from spiked in plasma samples. Sample SM1 to SM4 and SR1 to
SR4, are spiked samples with 10^5 copies/μl of the candidate miR-Seps 6.

Table 8B. Absolute quantification of endogenous miRNA in non-spiked RNA eluates.

Non-spiked RNA elutes Mean Cq ± SD Log concentration
(n=18)
NSM1 31.98±1.49 1.13E+03
NSM2 27.49±0.82 2.74E+04
NSM3 30.31±1.32 3.70E+03
NSM4 32.73±1.39 6.63E+02
NSM4 28.78±0.80 1.10E+04
NSM6 28.04±0.21 1.85E+04
NSM7 27.29±0.13 3.15E+04
NSM8 28.09±0.12 1.79E+04
NSM9 28.00±0.28 1.91E+04
NSR1 29.35±0.12 7.31E+03
NSR2 26.86±0.06 4.28E+04
NSR3* 32.79±0 6.35E+02
NSR4 26.66±0.39 4.93E+04
NSR5 27.59±0.14 2.55E+04
NSR6 28.02±0.53 1.88E+04
Page: 12
 NSR7 27.68±0.27 2.39E+04
NSR8 27.27±0.64 3.20E+04
NSR9 27.17±0.31 3.43E+04
* NSR3 only had one cq-value, as much, a standard deviation can not be calculated

Figure 5: Displays the standard curve after removing 10^3 and 10^2 copies of miR-seps 6 together
with absolute quantification of the unknown quantity of a the selected non-spiked and spiked elutes
mentioned in (Table 8A&B). A standard curve that is linear and has the formula y = -1,408ln(x) + 41,578.
Each synthetic miR-seps 6 replicate's average Cq value (y-axis) was determined and displayed against its
log copy number (x-axis) in the graph. R2 came in at 0.99. The red dots indicate NSM (n=9) samples,
whereas the green dots represent the quantified values of NSR (n=9).The SR10^5 (n=5) samples are
shown by the yellow dots, while the measurable SM10^5 (n=5) samples are represented by the grey dots.

A Shapiro-Wilk test was conducted to examine the normality distribution of the non-spiked
extractions for both the robotic (NSR) and manual (NSM) extractions. The test results indicate
that the robotic extractions are not normally distributed since they have a significance level
(p=0.954) whereas the manual extractions have a significance level (p=0.421). To determine if
there is a statistically significant difference between the manual and robotic methods of
extraction with regard to miR-seps 6 log copies/μl cDNA in specific, the median of the NSR (n=9)
and NSM (n=9) were compared (Table 8B). The median log copies/μl cDNA for the non-spiked
robotic samples (NSR) (n=9) was 2.55E+6 (IQR=2.55E+6), and the median concentration for the
non-spiked manual samples (NSM) (n=9) was 1.79 E+6 (IQR=2.08E+6) (Figure 6A). Also, the
Mann-Whitney U test was chosen to compare the two variables between the NSM and NSR in
order to determine whether there was a significant difference in the miR-seps 6 log copies/μl
cDNA obtained for each extraction technique. The p-value is 0.102, which means that there is a
significant difference between NSR and NSM samples for the distribution of the small RNA
concentration.
Also, the same test was run for the SM (n=4) and SR (n=4) samples (Table 8A). The median log
copies/μl cDNA for the spiked manual samples (SM) (n=4) was 4.93E+6 (IQR=4.23E+6), and the
median log copies/μl cDNA for the spiked robotic samples (SR) (n=4) was 1.96E+6
(IQR=4.23E+6) (Figure 6B). To test the normality distribution for both the robotic and the
manual extractions, a Shapiro-Wilk test was run. The test has shown that the robotic extractions
have a significance level (p=0.001), and the manual extractions have a significance level
(p=0.386) which means they are not normally distributed. As a results for the Mann-Whitney U
test, the p-value is 0.346, which means that there is a significant difference between NSR and
NSM samples for the distribution of the small RNA concentration.

Page: 13
A)

B)

Figure 6: The interquartile range (IQR) is displayed in each box. Each box displays the median of the data
as a horizontal black line. A 95% confidence interval can be seen on the vertical line. A) A comparison
between NSM (n=9) and NSR (n=9) extraction miR-seps 6 log copies/μl cDNA reaction. B) A comparison
between SR (n=4) and SM (n=4) extraction miR-seps 6 log copies/μl cDNA reaction. These extractions
were spiked with 10^5 copies/μl of miR-seps 6.

Melting curve analysis

During the two-tailed RT-qPCR (TATAA Biocenter) amplification procedure, all of the examined
non-spiked and spiked RNA eluates, both robotically and manually extracted, displayed a single
peak (data not shown). Additionally, the majority of the plates' negative controls (NTCs) did not
show any amplification (data not shown). The result of the melting curve for the candidate miR-
seps 6 cDNA templates is displayed in the following figure (Figure 7).

Page: 14
Figure 7: The melting curves of candidate miR-seps 6 cDNA templates and circulating miR-seps 6 cDNA.

Discussion
The sepsis diagnosis techniques used today lack the specificity required for a correct diagnosis
and takes too long time. Despite being a frequently used indicator of serious disease, CRP lacks
bacterial infection specificity and is shown to increase in the majority of other types of
inflammation (Duncan et al., 2021). Sepsis patients would live longer and less antibiotics would
be used unnecessarily if the condition could be diagnosed earlier and more effectively (Busch &
Kadri, 2020).
In this study, the miRNeasy® Serum/Plasma Advanced Kit (Qiagen) was chosen because it
would produce high-quality small RNA results, which are crucial in this research. The
miRNeasy® Serum/Plasma Advanced Kit (Qiagen) was found to be one of the top three kits as a
result of research conducted by Wright et al., (2020). With the same RNA yield as well as higher
quality, the kit is an alternative to the miRNeasy Serum/Plasma Kit. This is due to the fact that
miRNeasy Serum/Plasma Advanced Kit utilizes MinElute spin column technology without a
vacuum pump and is phenol-free. Usually, phenol residues degrade the extracted RNA's quality
(Wright et al.,2020). Phenol contamination might cause the actual concentration in extracted
samples to be underestimated or overestimated, which will have an adverse effect on the
findings of RT-qPCR (Unger et al., 2019). Nordén (2020), who had expertise evaluating many
miRNA isolation kits, discovered that the miRNeasy Serum/Plasma Advanced Kit had slightly
greater miRNA concentrations than the miRNeasy Serum/Plasma Kit, which led to improved
qPCR amplification results. Although the Serum/Plasma Advanced kit performed worse for
miRNA identification in frozen samples than in fresh plasma samples, this may have been since
the frozen samples had a lower cDNA content (Wright et al., 2020) . To minimize the impact of
technical variation, great care was taken to ensure that samples were handled similarly with
regard to RNA extraction and sample preservation. In order to protect the integrity of the
miRNA contained in the sample by preventing degradation, plasma extracts have been stored at
-80 °C (Vaught, 2006). The protocol suggested incubating at 37 degrees in a water bath until

Page: 15
samples are fully dissolved and salts are dissolved in the miRNeasy Serum/ Plasma Advanced
Kit Handbook 2021. The plasma sample, have been defrosted on ice and then incubated in the
VWB 2 (VWR) 37°C water bath for 20 seconds. Following that, 10 samples in total 100 μL each
were taken from the thawed plasma tubes and placed in Eppendorf 1.5 ml microcentrifuge
tubes. This procedure was used in this study to prevent contamination and ensure similar
plasma sample conditions.

Quantity and purity analysis

Small RNA concentrations for spiked samples maually extracted the SM(n=4) (Appendix E) and
SR10^5 copies/μl (n=1), SR10^6 copies/μl (n=1) and SR10^7 copies/μl (n=1) (Appendix D),
were too low to be measured by the Qubit 4.0 (Qiagen). Concentrations below the limit of
detection (LOD) are necessary to determine whether there is a difference between the
extraction efficiency of manual and robotic extraction since these RNA extractions that produced
a "too low to measure" could still include RNA. For statistical analysis, the undiscovered values
were changed out for a constant (Table 5). The LOD for the Qubit microRNA assay is 0.5 ng/μl
per sample (Qubit™ microRNA assay kits user guide, 2023). Therefore, it was decided to give the
extractions with concentration values that were too low to quantify
a 0.5 ng/μl concentration value (Qubit™ microRNA assay kits user guide, 2023). The LOD in this
instance was 0.5 ng/ μl due to the fact that 1 μl of the eluate was utilized as the input volume for
each experiment. To determine how much the data varied from the mean, the standard deviation
was calculated (Table 5). A standard deviation that is near to zero implies that the data was
accurate and close to the mean, although a high SD indicates that the data were not precise
(Darling, 2022). Thus, two-tailed RT-qPCR (TATAA Biocenter) was employed in the downstream
application using these eluates to successfully identify and quantify miRNAs. Generally, non-
spiked RNA eluates were more detectable than the spiked RNA eluates. The raw data for these
extractions are mentioned in (Appendix A and B). However, a statistical study was done to
compare which extraction method produced the highest concentration of the small RNA.
According to the analysis, the robotic extraction method produced a greater median
concentration than the other extraction method (Figure 2A). This does not imply, that the
manual extraction did not produce enough miRNA for further applications that will be required
for the two-tailed RT-qPCR to quantify it. For the non-spiked manual samples NSM (n=9), the
median of the small RNA concentration was 0.78 ng/μl (IQR=2.44) while for the non-spiked
robotic samples NSR (n=9), it was 4.94 ng/μl (IQR=0.36). These results of the concentration fall
in range concentrations were observed by Lindeberg (2020), who similarly used the QIAcube®
(Qiagen) and the miRNeasy serum/plasma advanced kit (Qiagen), with manual extraction
results of 0.69 ng/μl (SD±0.09) and robotic results of 1.79 ng/μl (SD±0.69). However,
Groenewald (2022) found values of 0.43 ng/μL (IQR=0.36) for manual extraction and 0.42
ng/μL (IQR=0.15) for robotic extraction, which is different from the observed extraction
concentrations. The quantification of RNA concentration in plasma samples is a fundamental
step in many molecular biology studies and diagnostic applications. However, it is well-known
that RNA concentrations can vary between different plasma samples. This variability can arise
from various factors, including biological variability, sample handling procedures, contaminants,
and extraction methods. Understanding and addressing these sources of variability are essential
for accurate and reliable RNA concentration measurements (Chomczynski et al., 2016). In
contrast, the IQR for the manual approach was 2.44, but the IQR for the robotic extraction
method was 0.36. This indicates that the small RNA concentration outcome had a repeatable
result, which resulted in a lower IQR for the robotic extraction method.
The Ds-11 spectrophotometer(Denovix) is more appropriate for demonstrating purity than
quantity since it is not designed to identify miRNAs but instead identifies other nucleotides,
Page: 16
proteins, or particles (Das Gupta et al., 2020). The manufacturer's website provides an
explanation of the 260/280 Ratio. The purity of DNA and RNA is determined by comparing the
absorbance at 260 nm and 280 nm. In general, RNA is considered to be "pure" with a ratio of
around 2.0. ‘
Negative findings from Denovix at the 260/280 absorbance rate indicate no difference between
the blank sample and the measured sample, whereas lower values indicate the presence of
proteins or additional contaminants (Lucena-Aguilar et al., 2016). The outcomes (Table 5) fall
below what was expected. This may be a sign of the presence of pollutants. On the other hand,
even if the procedure specifies that 1 μl would be enough, it may be worthwhile to test the
samples once more using up to 2 μl in order to be sure that no pipetting mistakes contributed to
the results. In addition, if the sample is at or below the detection limit, Nanodrop cannot
measure it effectively. To determine which method of extraction provided total RNA with the
best purity, a statistical analysis was conducted. The research showed that compared to the
other extraction technique, the robotic extraction approach provided a higher median
concentration (Figure 2B). The median A260/A280 ratio for NSR samples (n=9) was 1.48
(IQR=0.15), whereas it was 1.21 (IQR=0.90) for NSM samples (n=9).

The amount of isopropanol used and the centrifuge speeds are two examples of variables that
may vary between manual and robotic extraction and cause the A260/A280 ratio to differ
between the two methods. The QIAcube® (Qiagen) follows to a protocol unique to the miRNeasy
serum/plasma advanced kit (Qiagen), which requires a 200 μL serum/plasma starting volume.
230 μLof supernatant will thus be removed and added to the processing equipment, according to
this assumption. For this step, only 200 μL of plasma samples were placed into the QIAcube.
Thus, 230 μL of isopropanol must be added to achieve the isopropanol ratio of 1:1. Which
suggests that the robotically extracted sample now contains more isopropanol. The QIAcube®
adds 230 μL of isopropanol to the plasma sample. Given that it has been demonstrated that
differences in the isopropanol ratio during nucleic acid precipitation leads in varying miRNA
isolation efficiencies, this may have an impact on extraction efficiency. The QIAcube® has a
relative centrifugal force (RCF) limit of 12000, which is lower than the centrifuge used for
manual extraction, which has a maximum RCF of 14100 (QIAcube® user handbook, 2018). The
short RNA extraction efficiencies resulting from extractions where various RCF were applied
during manual extraction should have been empirically assessed, which was not done in this
study.

Hands on time and turn around time analysis

The non-spiked plasma extractions are the ones for which the time specified in (Table 6) was
measured. These were carried out following the spiked-in plasma extractions. The intention was
to acquire knowledge of the process and measure a time as near as feasible to a skilled
laboratory worker conducting comparable extractions in a clinical laboratory. (Table 6) shows
that manual extractions took longer time to complete than QIAcube-performed extractions in
terms of hands-on time. Since the samples are handled and touched less during brief hands-on
periods, there is a lower chance of cross-contamination. To estimate how long the extraction
procedure will take, however, the turnaround time is crucial. It is reasonable to assume that
QIAcube, especially when operating with a high flow of samples, might be an appropriate
solution to save effort, time, and complete many activities throughout its operation.

Standard curve and absolute quantification analysis

Absolute quantification of miRNAs in non-spiked and spiked RNA eluates was done after

Page: 17
optimization of the two-tailed RT-qPCR method. A standard curve was evaluated and improved
in order to improve the two-tailed RT-qPCR technology (Bio-Rad labs, 2006). The correlation
coefficient R2 should be greater than or equal to 0.98 for an optimal linear standard curve.
According to Bio-Rad labs (2006), the findings should have a high amplification efficiency (E)
ranging from 90% to 110% and be clearly consistent among duplicate reactions. (Table 7)
indicates that not all of the criteria had been met, but by eliminating 10^3 and 10^2 copies of
candidate miR-seps 6 (Integrated DNA Technologies), the standard curve optimization
was reached(Figure 4). The effectiveness of the PCR reaction is gauged by the slope of the
standard curve. For ten fold dilution, it should ideally be -3.3 (Svec et al., 2015). The R2 value did
not go over 0.99. However, because the efficiency was 103.3%, the standard curve optimization
results were relevant. QPCR efficiency is often the associated metric for the robustness and
accuracy of qPCR experiments (Svec et al., 2015). Using the improved standard curve, the
number of unknown copies of the miRNAs in the non-spiked and spiked samples was calculated
(Tables 8A and 8B). The two-tailed RT-qPCR approach was effective in detecting every miRNA
present in non-spiked and spiked RNA eluates. One outlier was determined while a statistical
analysis was run to check the difference between the robotic extractions and the manual. The
quantification of all the spike-in RNA eluates are detected (Figure 6B). The melting curves did
not show any indications of primer-dimers or secondary amplifications, which further
demonstrated how accurate and specific the two-tailed primers were (data not shown). With a
few minor exceptions, almost all of the miRNA copy numbers found in non-spiked and spiked
RNA eluates fell between copy numbers 10^2 and 10^4, according to (Tables 8A and 8B). Since
the plasma utilized in this research came from different adult donors, the variance may be
caused by particular variations in endogenous miRNAs across donors.

Absolute quantification of miR-seps 6 in non-spiked and spiked elutiones

The copy concentration of all miRNAs in spiked RNA eluates and the copy concentration of
endogenous miRNAs in non-spiked RNA eluates were calculated using absolute quantification
and the optimized standard curve, respectively (Tables 8A and 8B). Using the improved
standard curve (Figure 4B), unknown copies of the non-spiked and spiked miRNAs were
quantified (Figure 5). The two-tailed RT-qPCR approach was successful in detecting the majority
of the miRNAs in both non-spiked and spiked RNA eluates. Quantification was done on all of the
spiking tested RNA eluates. With a few minor exceptions, all of the miRNA copy measurements
found in NS-S RNA eluates were almost identical to those expected (Table 8A and 8B). Since the
plasma utilized in this research came from separate adult donors, the variance may be caused by
the unique changes in endogenous miRNAs across donors. Three samples of the NSM identified
themselves as outliers (Figure 5). The two-tailed RT-qPCR technology's ability to detect
endogenous miRNAs even at low copy quantities was promising. (Figure 5) illustrates how these
samples lie between 10^3 and 10^2. All of the 18 non-spiked samples that performed two-tailed
RT-qPCR demonstrated measurable findings from amplification. Nine of the measurable findings
came from samples that were manually extracted, and nine came from samples that were
robotically extracted. It may be concluded from the observed amplification that the test has the
ability to detect and amplify miR-seps 6. For the manual extraction approach, every measurable
non-spiked eluate generated a Cq value ranging from 27.29 to 32.73 (Appendix A; Table 2). The
robotic method's Cq value for the measurable non-spiked eluates varied from 26.66 to 32.79
(Appendix B; Table 2).
To determine the difference between the manual and the robotic method for the spiked and non-
spiked samples a statistical test was done. Both the non-spiked samples and the spiked samples
showed a substantial variation in the cq value (Figures 6A and 6B). However, the non-spiked
robotic samples (NSR) (n=9) had a median log copies/μl cDNA of 2.55E+6 (IQR=2.55E+6) and

Page: 18
the non-spiked manual samples (NSM) (n=9) had a median concentration of 1.79 E+6
(IQR=2.08E+6) (Figure 6A). In addition to being able to identify the cq-values for the samples
extracted using the robotic approach and demonstrating how it differs from manual extractions,
the two-tailed RT-qPCR method has been demonstrated to be successful for amplifying
circulating miR-seps 6 derived from 100 μl of human blood plasma. In order to assess the
differences in cq-values between robotic and manual extractions and prevent any potential
constraints brought on by variance, the identical plasma samples were planned to be utilized in
these extractions.

Melting curve
The RT, forward, and reverse primers displayed strong specificity, as seen by the well-defined
peaks between 76 and 80 °C that were evident in the melting curves produced from synthetic
miR-seps 6 (Figure 7). A miRNA standard curve generated by qPCR occasionally has two peaks
on the melting curve. Numerous causes can be implicated in this phenomenon. The first
possibility is that primer dimers are the cause. Short, non-specific products are created when the
qPCR primers anneal to one another rather than the target miRNA. Because primer-dimers often
have lower melting points than the particular product, there is an extra peak at a lower
temperature. But the melting curve result (Figure 7) reveals a little peak before at temperatures
between 60 and 70 degrees, which may still indicate a primer dimer. The NTC control, which
was performed concurrently, revealed amplification in two out of six wells (data not show).

Ethical consideration
MicroRNA studies as biomarkers for sepsis using plasma from healthy donors may not have
direct and immediate environmental benefits, but they can contribute to a more sustainable
healthcare system and indirectly support environmental well-being. One way in which this
research can be environmentally advantageous is through a reduced need for invasive
diagnostic procedures. By developing accurate and reliable microRNA biomarkers for sepsis, it
may be possible to minimize the reliance on invasive tests that require additional resources and
generate medical waste. This could lead to a reduction in the environmental impact associated
with these procedures. Furthermore, the identification of specific microRNA biomarkers for
sepsis can pave the way for precision medicine and targeted therapies. By tailoring treatments
based on individual biomarker profiles, precision medicine aims to optimize therapeutic
outcomes while minimizing unnecessary medication use. This approach can reduce the overall
environmental burden associated with pharmaceutical production, usage, and disposal. Early
detection of sepsis through microRNA biomarkers can also contribute to improved disease
management and resource allocation. By identifying sepsis cases at an early stage, healthcare
interventions can be initiated promptly, potentially preventing unnecessary hospitalizations and
reducing the length of hospital stays. This streamlined approach minimizes the environmental
impact associated with prolonged medical care. Additionally, microRNA research in sepsis
contributes to the advancement of scientific knowledge and innovation. The understanding
gained from these studies can be applied to other areas of medicine and research, leading to
further advancements in diagnostics and therapies. Such progress may have direct
environmental benefits in other domains of healthcare and medicine. While the immediate
environmental impact of microRNA sepsis research may be limited, the potential improvements
in healthcare efficiency, targeted therapies, and reduced reliance on invasive procedures can
collectively contribute to a more sustainable healthcare system. Moreover, the scientific
knowledge gained from these studies can have broader implications for future research and
innovations that may directly benefit the environment in various areas of healthcare and
beyond.

Page: 19
Conclusion
Three scientific questions were looked into in this study. It has been established that there are
variations between carrying out the extraction using the robotic approach and the manual way.
However, in this investigation, all samples, including those from the robotic approach, had lower
purity ratios than anticipated. In terms of concentration, the robotic technique of extraction
seems to have produced better outcomes. Even though the two-tailed RT-qPCR approach could
also identify a low copy number in the non-spiked plasma samples, it has been demonstrated to
be successful in amplifying circulating miR-seps 6 obtained from 100 μl of human blood plasma.
The melt curve showed a single product, which correlates with excellent specificity, and a linear
standard curve made from synthetic miR-seps 6 generated the best amplification efficiency.
However, it is clear that optimization is necessary before the test is used in a clinical setting.

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Appendix A
Table 1. Concentrations absorbance measurements and purity ratios. Raw data collected from n
on spiked samples manually extracted.
Sample Concentration A260 260/230 260/280 Small RNA conc
ng/μl entration by Q
ubit (ng/μl)
NSM1 11.25 0.28 0.20 0.89 0.80
18.54 0.46 0.15 1.21 0.78
17.18 0.43 0.10 1.21 0.74
NSM2 7.05 0.18 0.11 0.79 0.70
6.81 0.17 0.12 0.83 0.68
9.27 0.23 0.13 0.94 0.66
NSM3 8.40 0.21 0.17 0.98 0.68
5.37 0.13 0.11 0.66 0.66
7.92 0.20 0.14 0.94 0.64
NSM4 3.20 0.08 0.16 0.91 0.76
4.93 0.12 0.20 1.10 0.72
5.50 0.14 0.19 1.02 0.74
NSM5 4.39 0.11 0.04 0.60 0.74
7.07 0.18 0.06 0.90 0.74
8.30 0.21 0.06 1.0 0.72
NSM6 5.38 0.13 0.02 2.25 2.20
7.28 0.18 0.03 1.71 2.02
6.80 0.17 0.03 1.84 1.88
NSM7 8.16 0.20 0.19 1.88 3.66
9.07 0.23 0.20 1.37 3.56

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 9.13 0.23 0.20 1.39 3.40
NSM8 5.62 0.14 0.30 1.86 3.16
6.60 0.16 0.31 1.81 3.00
8.93 0.22 0.39 2.62 2.88
NSM9 4.23 0.11 0.04 1.29 4.18
6.21 0.16 0.06 1.57 3.98
7.85 0.20 0.07 2.11 3.82

Table 2. The average of the Cq-value for the NSM qPCR quantification
Sample Mean of the Cq-value
NSM1 31.98
NSM2 27.49
NSM3 30.31
NSM4 32.73
NSM5 28.78
NSM6 28.04
NSM7 27.29
NSM8 28.09
NSM9 28.00

Appendix B
Table 1. Concentrations absorbance measurements and purity ratios. Raw data collected from n
on spiked samples robotic extracted.
Sample Concentration A260 A260/A230 A260/280 Small RNA conc
ng/μl entration by Q
ubit (ng/μl)
NSR1 13.03 0.33 0.22 1.46 4.54
18.23 0.46 0.27 1.49 4.54
13.99 0.35 0.22 1.70 4.52
NSR2 15.31 0.38 0.23 1.42 5.02
13.64 0.34 0.21 1.40 4.98
12.18 0.30 0.18 1.57 4.90
NSR3 18.82 0.47 0.38 1.5 5.04
22.47 0.56 0.41 1.54 4.98
17.86 0.45 0.39 1.48 4.94
NSR4 5.16 0.13 0.17 1.71 5.14
4.65 0.12 0.15 1.48 5.10
6.91 0.17 0.19 1.60 5.02
NSR5 21.56 0.54 0.21 1.60 5.08
26.15 0.65 0.24 1.58 4.92
20.97 0.52 0.19 1.55 4.82
NSR6 15.49 0.39 0.21 1.35 4.84
9.49 0.24 0.14 1.19 4.56
11.53 0.29 0.16 1.43 4.52
NSR7 9.79 0.25 0.16 1.44 5.02
9.98 0.25 0.16 1.42 4.96
9.41 0.24 0.15 1.41 4.90
NSR8 7.06 0.18 0.11 1.33 5.28
9.08 0.23 0.14 1.42 5.12
8.17 0.20 0.12 1.56 5.06
NSR9 8.73 0.22 0.19 1.37 4.72
4.69 0.12 0.11 0.96 4.68
6.06 0.15 0.14 1.52 4.64

Page: 24
Table 2. The average of the Cq-value for the NSR qPCR quantification
Sample Mean of the Cq-value
NSR1 29.35
NSR2 26.86
NSR3 32.79
NSR4 26.66
NSR5 27.59
NSR6 28.02
NSR7 27.68
NSR8 27.27
NSR9 27.17

Appendix C
Table 1. Concentrations absorbance measurements and purity ratios. Raw data collected from sp
iked plasma samples with different copy numbers. These are for the manually extracted
samples.
Sample Concentration A260 A260/A230 A260/280 Small RNA conc
ng/μl entration by Q
ubit (ng/μl)
SM10^5 4.62 0.12 0.09 0.80 1.14
3.33 0.08 0.06 1.26 0.88
3.76 0.09 0.07 0.86 0.66
SM10^6 4.74 0.12 0.16 1.02 0.84
8.84 0.22 0.29 1.60 0.72
8.45 0.21 0.28 1.18 0.80
SM10^7 7.35 0.18 0.20 1.33 1.53
6.29 0.16 0.17 0.95 1.40
7.36 0.18 0.19 1.50 1.29

Appendix D
Table 1. Concentrations absorbance measurements and purity ratios. Raw data collected from sp
iked plasma samples with different copy numbers. These are for the robotic extracted samples.
Sample Concentration A260 A260/A230 A260/280 Small RNA conc
ng/μl entration by Q
ubit (ng/μl)
SR10^5 2.81 0.07 0.10 2.98 Out of range
2.86 0.07 0.09 1.24
7.52 0.19 0.19 1.38
SR10^6 8.73 0.22 0.17 1.63 Out of range
8.38 0.21 0.16 1.45
7.90 0.20 0.14 1.67
SR10^7 18.30 0.46 0.35 1.66 Out of range
23.86 0.60 0.39 1.58
17.47 0.44 0.32 1.74

Table 2. The average of the Cq-value for the spiked plasma samples with different copy numbers
qPCR quantification.
Sample Mean of the Cq-value
SM10^5 26.46
SM10^6 24.44
SM10^7 21.79
Page: 25
 SR10^5 25.68
SR10^6 23.76
SR10^7 24.91

Appendix E
Table 1. Concentrations absorbance measurements and purity ratios. Raw data collected from sp
iked plasma samples with 10^5 copies/ul. Following data are from manually extracted samples.
Sample Concentration A260 A260/A230 A260/280 Small RNA con
ng/μl centration by
Qubit (ng/μl)
SM1 7.64 0.19 0.35 1.40
8.30 0.21 0.34 1.29 Out of range
6.62 0.17 0.31 1.27
SM2 3.91 0.10 0.04 1.70
6.77 0.17 0.07 1.53 Out of range
4.90 0.12 0.05 1.52
SM3 3.329 0.08 0.04 1.13
4.03 0.10 0.05 1.16 Out of range
5.15 0.13 0.06 1.22
SM4 4.83 0.12 0.12 1.28
5.80 0.16 0.13 1.42 Out of range
5.86 0.15 0.13 1.47

Table 2. The average of the Cq-value for the SM qPCR quantification.

Sample Mean of the Cq-value
SM1 26.19
SM2 27.81
SM3 26.66
SM4 27.97

Table 3. Concentrations absorbance measurements and purity ratios. Raw data collected from sp
iked plasma samples with 10^5 copies/ul. Following data are from robotic extracted samples.
Sample Concentration A260 A260/A230 A260/280 Small RNA con
ng/μl centration by
Qubit (ng/μl)
SR1 9.86 0.25 0.24 1.38 0.52
10.09 0.25 0.23 1.38 0.62
9.68 0.24 0.21 1.37 0.52
SR2 11.65 0.29 0.28 1.06 0.96
12.03 0.30 0.29 1.15 0.86
11.81 0.30 0.29 1.25 0.78
SR3 14.55 0.36 0.13 1.49 1.19
14.64 0.37 0.13 1.40 1.14
15.77 0.39 0.13 1.46 1.04
SR4 6.48 0.16 0.11 1.19 1.65
8.83 0.22 0.14 1.26 1.41
9.95 0.25 0.14 1.27 1.34

Table 4. The average of the Cq-value for the SR qPCR quantification.

Page: 26
 Sample Mean of the Cq-value
SR1 28.06
SR2 27.77
SR3 28.06
SR4 27.96

Table 5. Absolute quantification of endogenous miRNA in the spiked plasma samples with differe
nt copy numbers.
Spiked RNA elutes Mean Cq ± SD Log concentration
With 10^5
SM10^5 26.46±0.11 5.69E+04
SR10^5 25.68±0.43 9.89E+04
SM10^6 24.44±0.28 2.39E+05
SR10^6 23.76±0.26 3.87E+05
SM10^7 21.79±0.25 1.57E+06
SR10^7 24.91±0.07 1.71E+05
SM1 26.19±0.02 6.89E+04
SM2 27.81±0.14 2.18E+04
SM3 26.66±0.11 4.93E+04
SM4 27.97±0.12 1.95E+04
SR1 28.06±0.28 1.83E+04
SR2 27.77±0.26 2.24E+04
SR3 28.06±0.16 1.83E+04
SR4 27.96±0.16 1.96E+04

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