RNA function, diversity, and key

characteristics

1 2 3 4

4
The role of RNA in Central Dogma: Identifying the Transcriptome

5
The role of RNA in Central Dogma: RNA Biotypes

~80% of mammalian genome is actively transcribed Mammalian Cell Nucleus

Genome
Non-coding RNA (~90% of the transcriptome)

ncRNA

Transcriptome
transcripts that do not code for proteins, but are
intimately involved with regulation of protein-encoding
transcripts
6
The role of RNA in Central Dogma: RNA Biotypes

~80% of mammalian genome is actively transcribed Mammalian Cell Nucleus

Genome
Non-coding RNA (~90% of the transcriptome)

ncRNA

Transcriptome
rRNA constitutes the majority of ncRNA and is used
often as a control to determine successful RNA
extraction
7
The role of RNA in Central Dogma: RNA Biotypes

~80% of mammalian genome is actively transcribed Mammalian Cell Nucleus

Genome
Coding RNA (~5% of the transcriptome)

ncRNA

transcripts that code for

protein products

mRNA

Transcriptome

8
The role of RNA in Central Dogma: RNA Function
Transcription dsDNA
Translation

RNA

DNA
9
The role of RNA in Central Dogma: RNA Function
Transcription Translation

ncRNA regulate gene expression at multiple phases

Pre-mRNA
processing

Mature mRNA

10
The role of RNA in Central Dogma: RNA Characteristics

Mature mRNA ncRNA

• Contain coding regions (exon sequences)

• 5’ cap and 3’ Poly A tail (untranslated regions) • A very diverse group of RNA with
wide-ranging characteristics
• Short half-life (unstable; cellular lifespan of minutes to days) • Variable size (19nt – 100Kb)

• May contain modified sequence 3’/5’caps

• Short half-life (unstable)

11
RNA Isolation: The importance of pure, intact
extract

1 2 3 4

12
RNA isolation: From RNA to cDNA Amplification

13
RNA isolation: From RNA to cDNA Amplification

cDNA
Sample RNA Isolation cDNA Synthesis

RNA RNA
Reverse Transcriptase

GENE X levels

14
RNA isolation: From RNA to cDNA Amplification

Sample RNA Isolation cDNA Synthesis

cDNA Synthesis

RNA RNA

GENE X levels

15
RNA Isolation: Importance of minimizing impurities
Successful qPCR begins with a pure extract of RNA molecules…

RT-PCR/PCR inhibitors
Reverse Transcription cDNA
other impurities

protein
RNA
RNA
Reverse
DNA Transcriptase

PCR Amplification

Sample RNA Extract

Polymerase

16
RNA Isolation: Importance of minimizing impurities
Successful qPCR begins with a pure extract of RNA molecules…

RT-PCR/PCR inhibitors Impure Pure

other impurities
RNA extract purity scale
protein
RNA
RNA

DNA

PCR Amplification

Amplification cycles (Cq)

Sample RNA Extract

17
RNA Isolation: Importance of minimizing impurities
Successful qPCR begins with a pure extract of RNA molecules…

RT-PCR/PCR inhibitors Impure Pure

other impurities
RNA extract purity scale
protein
RNA
RNA

DNA

PCR Amplification

Amplification cycles (Cq)

Sample RNA Extract

18
RNA Isolation: Importance of minimizing impurities
Successful qPCR begins with a pure extract of RNA molecules…

RT-PCR/PCR inhibitors Impure Pure

other impurities
RNA extract purity scale
protein
RNA
RNA

DNA

PCR Amplification

Amplification cycles (Cq)

Sample RNA Extract

19
RNA Isolation: Importance of minimizing impurities
Successful qPCR begins with a pure extract of RNA molecules…

Reverse Transcription cDNA

other impurities

protein
RNA
RNA
Reverse
DNA Transcriptase

PCR Amplification

Sample RNA Extract

Polymerase

20
RNA Isolation: Importance of RNA integrity
Successful qPCR begins with a pure extract of RNA molecules…that are intact

Reverse Transcription cDNA

other impurities

protein
RNA
RNA ...low integrity
Reverse
DNA Transcriptase

PCR Amplification

Sample RNA Extract

Polymerase

21
RNA Isolation: Importance of RNA integrity
Successful qPCR begins with a pure extract of RNA molecules…that are intact
Intact Degraded

other impurities
RNA extract integrity
protein
RNA
RNA…intact

DNA

PCR Amplification

Amplification cycles (Cq)

Sample RNA Extract

22
RNA Isolation: Importance of RNA integrity
Successful qPCR begins with a pure extract of RNA molecules…that are intact
Intact Degraded

other impurities
RNA extract integrity
protein
RNA
RNA ...low integrity

DNA

PCR Amplification

Amplification cycles (Cq)

Sample RNA Extract

23
RNA Isolation: Importance of RNA integrity
Successful qPCR begins with a pure extract of RNA molecules…that are intact
Intact Degraded
cDNA
other impurities
RNA extract integrity
protein
RNA
RNA ...low integrity

DNA

PCR Amplification

Amplification cycles (Cq)

Sample RNA Extract

24
RNA Isolation: Biotypes and Extraction Kits

Thinking Ahead RNA Biotype Important Considerations

mRNA Use a kit that can perform “total RNA”

extraction

Which RNA biotype am I interested

in studying and which RNA extraction short-ncRNA/miRNA Avoid column purification steps based on
kit is best for isolating this biotype? size exclusion; seek out specialized short
-ncRNA or miRNA kits; do not rely on kits
that claim “total RNA” extraction

25
RNA Isolation: Sample Type Considerations

Thinking Ahead Sample Type Important Considerations

Tissue prepare RNA isolations with fresh tissue or flash

What is the source of my RNA
and how should I proceed
accordingly?
frozen tissue if possible; freeze thaw cycles will
degrade RNA; includes many cell types; represent a
reservoir rich in impurities/PCR inhibitors
Cells Assess the viability of your cells before harvesting;
start with the same number of cells per sample;
use the recommended number of cells for the
extraction kit
Blood Represents a reservoir low in cells/RNA; will need
large amounts of blood and good concentration
protocol to get enough RNA

26
RNA Isolation: Bio-Rad RNA Extraction Kits/Protocols

Thinking Ahead

What is the source of my RNA

and how should I proceed
accordingly?

27
RNA Isolation: Preserving RNA stability and reducing contamination
-80°C Freezer
Key considerations to keep RNA integrity high and contamination low
RNase-free solution
1. Store RNA samples at -80°C

2. Decontaminate all surfaces, pipettors

benchtops, glassware, and gel equipment -20°C freezer RNA sample
with RNase-free solutions (i.e. RNase Zap)

3. Wear appropriate PPE, including gloves,

lab coat, and safety glasses
Wear PPE
4. Avoid freeze-thaw cycles to RNA samples
Aerosol Tips

5. Make sure to implement DNAse, especially

if working with samples from organisms with
very small or no introns

6. Use aerosol-barrier tips

DNAse

28
Exploring methods to validate
extract quality and quantity

1 2 3 4

29
Extract Quality and Quantity: Knowing what you’re starting with

Before starting cDNA synthesis, it’s best to test your

RNA extract to define its…

…quantity: How much RNA is in your extract?

RNA Extract

…quality: What is the status of the RNA purity and integrity?

30
Extract Quality and Quantity: Methods to detect RNA quantity/quality

Spectrophotometry: UV Absorbance Method: The UV absorbance of diluted RNA sample(s) is measured at

230nm, 260nm and 280nm wavelengths.
UV Abs. 260nm/280nm Ratio

1.51 1.15 4.50

NanodropTM

1.47 4.00

31
Extract Quality and Quantity: Methods to detect RNA quantity/quality

Spectrophotometry: UV Absorbance Method: The UV absorbance of diluted RNA sample(s) is measured at

230nm, 260nm and 280nm wavelengths.

Quality assessment 1 (260nm/280nm):

• Pure RNA will read at 1.8-2.0
• Elevated readings suggest presence of proteins and/or
NanodropTM phenol

Quality assessment 2 (260nm/230nm):

• Pure RNA will read from 2.0-2.2
• Elevated readings suggest presence of carbohydrates and/or
EDTA

32
Extract Quality and Quantity: Methods to detect RNA quantity/quality

Spectrophotometry: UV Absorbance Method: The UV absorbance of diluted RNA sample(s) is measured at

230nm, 260nm and 280nm wavelengths.

Pros: spectrophotometers are readily available in most laboratories;

NanodropTM enables RNA quantitation with only 1-2 µL of sample; wide
dynamic range (2ng.ul – 12,000ng/ul); quick protocol (~30 sec)

Cons: The reading relies upon the purity of the samples; evidently,
contaminating substances with absorption at 260nm or 280nm
will affect the estimated OD reading.

33
Extract Quality and Quantity: Methods to detect RNA quantity/quality

Gel Electrophoresis: RNA integrity Method: An agarose gel acts as a matrix to contain and separate the
nucleic acid molecules (DNA/RNA). The gel is submerged in an
electrophoresis chamber with buffer that allows the flow of an
electric current.

- RNA Migration +
28S rRNA

UV Transillumination
18S rRNA

Gel (EtBr)

Running Buffer

34
Extract Quality and Quantity: Methods to detect RNA quantity/quality

Gel Electrophoresis: RNA integrity Method: An agarose gel acts as a matrix to contain and separate the
nucleic acid molecules (DNA/RNA). The gel is submerged in an
electrophoresis chamber with buffer that allows the flow of an
electric current.

Pros: Cost effective; most labs are equipped with gel electrophoresis
technology; easy to follow protocol

Cons: A normal procedure can take up to an hour to complete;

suggested amounts of RNA input for electrophoresis can
exceed 5ug

35
Extract Quality and Quantity: Methods to detect RNA quantity

Bioanalyzer InstrumentTM Method: Automated electrophoresis; combines microfluidics and a

proprietary algorithm to calculate a RIN number from the ratio
of the 18S to 28S ribosomal subunits

36
Extract Quality and Quantity: Methods to detect RNA quantity

Bioanalyzer InstrumentTM Method: Automated electrophoresis; combines microfluidics and a

proprietary algorithm to calculate a RIN number from the ratio
of the 18S to 28S ribosomal subunits

Pros: Easily determine the RNA integrity number (RIN) from total
RNA sample; uses little input of RNA (~5ul); calculates RNA
concentration of HMW RNA biotypes

Cons: Not always available in the lab space

37
Extract Quality and Quantity: Methods to detect RNA quantity/quality

QubitTM Fluorometer: Method: fluorescent dye emits signal when bound to the specific target
molecules (DNA or RNA) even in the presence of free
nucleotides and or degraded nucleic acids

Pros: RNA quantity, integrity, and quality can be rapidly measured

using highly sensitive Qubit assays even in RNA samples with
very low concentration

Cons: Not always available in the lab space

38
Extract Quality and Quantity: RNA input for cDNA synthesis

How much RNA is enough to produce good quality data?

If we assume the following…

✔ High quality RNA extract

✔ High enzyme performance (RT and Pol)
✔ Enough RNA extract for your given cDNA synthesis kit
…then we can make an educated guess based on the abundance of the target

39
Extract Quality and Quantity: RNA input for cDNA synthesis

How much RNA is enough to produce good quality data?

General guidance for RNA input:

RNA Extract Recommended Range of Input

Transcript Abundance Total RNA 10 pg – 5ug
Poly(A) RNA 10 pg – 0.5ug
Input RNA Extract Conc. Specific RNA 0.01 pg – 0.5ug

40
Extract Quality and Quantity: RNA input for cDNA synthesis

How much RNA is enough to produce good quality data?

✔ Reproducible C q values

Ultimately, it comes down to how your results look:

Examining the results
Consider the following RT-qPCR experiment

•Targeting PKG-1 mRNA (~160 bp), a gene

that encodes a glycolytic enzyme
•1-step process
•RNA input (2 conc.):
•100 ng/rxn
•100 pg/rxn
•20 technical replicates

12 34

41
Extract Quality and Quantity: RNA input for cDNA synthesis

How much RNA is enough to produce good quality data?

Ultimately, it comes down to how your results look:

✔ Logical difference between C q
values of different RNA inputs
Examining the results
Consider the following RT-qPCR experiment

•Targeting PKG-1 mRNA (~160 bp), a gene

that encodes a glycolytic enzyme
~10 cycles values
•1-step process
•RNA input (2 conc.):
•100 ng/rxn
•100 pg/rxn
•20 technical replicates

12 34

42
Extract Quality and Quantity: RNA input for cDNA synthesis

How much RNA is enough to produce good quality data?

✔C values aren’t outside the acceptable
q
range for RT-qPCR detection
Ultimately, it comes down to how your results look:
Examining the results
Consider the following RT-qPCR experiment

Questionable Ct values

Questionable Ct values
•Targeting PKG-1 mRNA (~160 bp), a gene
that encodes a glycolytic enzyme
•1-step process
•RNA input (2 conc.):
•100 ng/rxn
•100 pg/rxn
•20 technical replicates

12 34

43
cDNA Synthesis: RT process, enzymes,
and suitability for RNA biotypes

1 2 3 4

44
cDNA synthesis: From RNA to cDNA

45
cDNA synthesis: From RNA to cDNA

Sample RNA Isolation cDNA Synthesis

cDNA Synthesis

RNA RNA

GENE X levels

46
cDNA synthesis: 1-step vs. 2-step RT-qPCR
2-Step RT-qPCR

Step 1. Reverse Transcription/cDNA synthesis

Representative RNA isolate (mRNA)

Primers anneal Reverse Transcriptase binds

5’ 3’ 5’
RT
Primer cDNA
mRNA (oligo dT)
cDNA

cDNA extension Incorporation of dNTPs 3’ 5’

RT5’
Step 1.
cDNA
Tube 1
mRNA

Tube 1

47
cDNA synthesis: 1-step vs. 2-step RT-qPCR
2-Step RT-qPCR

Step 2. cDNA Amplification (PCR) 5’

3’ Representative cDNA (from mRNA)

cDNA

Primers/Probes anneal Polymerase binds 5’

3’
PCR Amplification
5’
P cDNA 3’
F Primer
cDNA PCR 5’ Probe
amp (specific)
DNA extension Incorporation of dNTPs
5’
Step 1. Step 2. 5’
3’ cDNA P
Tube 1 Tube 2 5’
3’

Tube 2

48
cDNA synthesis: 1-step vs. 2-step RT-qPCR
1-Step RT-qPCR

1. Primer 1. Primer/ 2. Polymerase

2. RT binding
Annealing Probe Annealing binding

Reverse Transcription PCR Amplification

Step1/Tube 1 Step 2/Tube 2

3. cDNA
3. Amplification
Synthesis

Both steps
Occur sequentially in 1 tube

49
cDNA synthesis: 1-step vs. 2-step RT-qPCR

1-Step 2-Step

cDNA
+
PCR
amp cDNA + PCR
amp

Pros: • Fast (eliminates benchtop work) • Can archive cDNA and use multiple times
• Decreases the chance of contamination for additional RT-qPCR rxns
• Allows for a pre-amp step when targeting
rare events

Cons: • Cannot reuse the cDNA for additional • Slower than 1-step
RT-qPCR rxns • More pipetting steps means increased risk
• Conditions in the chemical reaction are of contamination
not optimal for both RT and Pol

50
cDNA synthesis: Examining the properties of RTs

Not all RT enzymes are created equal 1) RT without nuclease activity

3’ 5’
RT RT RT
mRNA cDNA

Importance of RNase H+ activity in RT-qPCR 2) RT with nuclease (RNase H+) activity

1. Generates a 1:1 ratio of transcript to cDNA 3’ 5’

RT
2. Provides unbiased transcription amid high mRNA cDNA
background
3. Increases likelihood of transcribing rare
transcripts
4. Removal of RNA transcripts prevents
RNA-blocking of second strand synthesis

51
cDNA synthesis: Examining the properties of RTs

Not all RT enzymes are created equal 2) RT with nuclease (RNase H+) activity
RNA degradation
3’ F Primer 3’ 5’
RT RT RT 5’

mRNA cDNA

Importance of RNase H+ activity in RT-qPCR

1. Generates a 1:1 ratio of transcript to cDNA

2. Provides unbiased transcription amid high

background
3. Increases likelihood of transcribing rare
transcripts
4. Removal of RNA transcripts prevents
RNA-blocking of second strand synthesis

52
cDNA synthesis: Examining the properties of RTs

Not all RT enzymes are created equal 1) RT without nuclease activity

RNA degradation
3’ F Primer 3’ 5’
RT RT RT 5’
mRNA cDNA

Importance of RNase H+ activity in RT-qPCR Common RT’s with nuclease activity

1. Generates a 1:1 ratio of transcript to cDNA 1. Avian Myeloblastosis Virus (AMV) RT

2. Provides unbiased transcription amid high 2. Moloney Murine Leukemia Virus (MMLV) RT (Bio-Rad’s iScript Kits)
background
3. Human Immunodeficiency Virus (HIV) RT
3. Increases likelihood of transcribing rare
transcripts
4. Removal of RNA transcripts prevents
RNA-blocking of second strand synthesis

53
cDNA synthesis: Selecting the appropriate RT for RT-qPCR
Thinking Ahead
Which Reverse Transcriptase is
right for my RNA target?
54
Reverse Transcriptase Important Considerations
AMV high processivity for reverse transcribing longer
transcripts; low RNase H+ activity that will
amount to lower yields of cDNA
Best representation and yield
MMLV (iScript Kits) good thermostability (50°C limit); high level of
RNase H+ activity that will amount to higher
yields; good fidelity/accuracy (1 in 30,000 nts)
HIV high thermostability (>60°C); performs well with
GC-rich transcripts (>70%); low fidelity/accuracy
(1 in 2,000 nts)
cDNA synthesis: Identifying priming strategies

2-Step RT-qPCR: Priming strategies for generation of cDNA

Step 1. Reverse Transcription/cDNA synthesis

Representative RNA isolate (mRNA)

Priming Strategies Primers anneal Reverse Transcriptase binds

Oligo dT 3’ 5’
5’
mRNA RT
Primer
(oligo dT)

cDNA extension Incorporation of dNTPs 3’ 5’

5’
3’
mRNA cDNA

55
cDNA synthesis: Identifying priming strategies

2-Step RT-qPCR: Priming strategies for generation of cDNA

Step 1. Reverse Transcription/cDNA synthesis

Representative RNA isolate (mRNA)

Priming Strategies Primers anneal Reverse Transcriptase binds

Oligo dT 3’
5’
Random Hexamer RT mRNA RT
5’
5’ Primer
Primer (Random
(Random Hexamer)
Hexamer)
cDNA extension Incorporation of dNTPs 3’ 5’
5’
3’
mRNA cDNA

56
cDNA synthesis: Identifying priming strategies

2-Step RT-qPCR: Priming strategies for generation of cDNA

Step 1. Reverse Transcription/cDNA synthesis

Representative RNA isolate (mRNA)

Priming Strategies Primers anneal Reverse Transcriptase binds

Oligo dT 3’
5’
Random Hexamer mRNA RT
5’
Gene Specific Primer
(Gene
Specific)
cDNA extension Incorporation of dNTPs 3’ 5’
5’
3’
mRNA cDNA

57
cDNA synthesis: Selecting the appropriate RT for RT-qPCR

A mixture of oligos and random primers can often be the best strategy if not
Thinking Ahead Primming Strategy Important
using Considerations
gene specific primers

Oligo dT Only captures processed mRNA; initiates the

synthesis preferentially at the 3’ end of the RNA
fragment, which can lead to pre-mature
Which priming strategy is termination if the transcript has increased 2*
right for my RNA target(s)? structure
Random Hexamer Can opt for small oligos increases chance of
complete coverage; can lead to an
overestimation of copy number due to internal
Priming; may represent a 3’to 5’ bias

Gene Specific Use if you know what your target of interest is;
use for 1-step RT-qPCR; best option for the least
amount of steps/time

58
cDNA synthesis: Selecting a priming strategy for 2-step RT-qPCR

59