Reports

An optimized TRIzol-based method for isolating

RNA from adipose tissue
Hongwei Zhang‡ ,1,2 , Yaoming Liu‡ ,1 , Bingcheng Yu2 & Rong Lu*,1
1
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology & Visual Science,
Guangzhou, 510060, China; 2 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510060, China; *Author for correspondence: Tel.: +86 138 2645 6581;
lurong@gzzoc.com; ‡ Authors contributed equally

BioTechniques 74: 203–209 (May 2023) 10.2144/btn-2022-0120

First draft submitted: 29 November 2022; Accepted for publication: 19 April 2023; Published online: 26 May 2023

ABSTRACT
High-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is difficult. Many studies have made
efforts to optimize methods for isolating RNA from adipose tissue through combinations of column-based kits and phenol-chloroform methods,
or through in-house protocols. However, the considerable complexity of these protocols and the various kits/materials required hamper their
wide use. Herein, we describe an optimized protocol based on TRIzol reagent, which is the most accessible ready-to-use reagent for nucleic
acid and/or protein isolation in laboratories. This article provides a step-by-step protocol yielding sufficient and qualified RNA from lipid-rich
specimens for downstream applications.

METHOD SUMMARY
A total of 22 human orbital fat specimens of the same quality were split into two groups, classic and optimized. RNA were extracted by the
traditional method and the optimized method, respectively, using TRIzol™ Reagent (Invitrogen, CA, USA). The yield, integrity and purity of the
two groups of RNA were compared. The optimized steps include, but are not limited to, soaking the tissue clipped down in liquid nitrogen, using
a low-temperature homogenizer to homogenize the sample, clearing the fatty layer after centrifuge and changing the time and temperature of
some centrifuge steps.

TWEETABLE ABSTRACT
An optimized protocol based on TRIzol for RNA extraction from human fat is described.

GRAPHIC ABSTRACT

Centrifuge the screws

Centrifuge the tubes

Remove the fatty layer

Transfer the cleared supernatant Add chloroform and mix
Add 1ml TRIzol reagent

Agitate the samples

Centrifuge the tubes

Centrifuge the tubes

Wash RNA pellet with 75% ethanol

Discard the supernatant and dissolve RNA Transfer the colorless aqueous phase
Add precooled isopropanol

Optimized protocol yielding RNA from lipid-rich specimens.

KEYWORDS:
Adipose tissue • RNA integrity • RNA isolation • TRIzol

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Table 1. Concentration and quality of RNA extracted in two groups.

Patient id Method Concentration (ng/ul) A260/A280 A260/A230 RIN
1 Classic 354 1.85 1.09 4.3
2 Classic 521 1.79 1.86 4.5
3 Classic 336 1.75 1.34 4.5
4 Classic 720 1.75 1.84 5.1
5 Classic 232 1.75 2.20 5.0
6 Classic 297 1.71 1.55 4.0
7 Classic 628 1.72 2.01 3.3
8 Classic 148 1.78 1.55 3.4
9 Classic 202 1.65 1.67 4.7
10 Classic 78 1.79 1.97 3.9
11 Classic 655 2.10 1.85 4.4
12 Optimized 320 2.01 2.07 6.2
13 Optimized 298 1.99 2.19 6.1
14 Optimized 72 2.02 2.09 7.1
15 Optimized 140 1.97 2.22 5.7
16 Optimized 240 1.97 2.19 5.6
17 Optimized 226 2.02 2.06 6.7
18 Optimized 332 1.94 2.19 5.9
19 Optimized 258 1.94 2.22 6.5
20 Optimized 224 2.01 1.99 7.3
21 Optimized 1889 2.02 2.01 7.1
22 Optimized 154 2.02 2.06 6.5
RIN: RNA Integrity Number.

Table 2. Comparison of RNA yield and quality between two groups.

Parameter Classic Optimized p-value
Concentration (ng/ul) 379.18 ± 219.25 377.54 ± 507.51 0.2505
A260/A280 1.78 ± 0.12 1.99 ± 0.032 0.001087
A260/A230 1.72 ± 0.32 2.12 ± 0.086 0.0009994
RNA Integrity Number 4.28 ± 0.59 6.43 ± 0.58 7.005e-05

The target tissue of many diseases is lipid-rich, such as the cortex in brain disease and orbital fat in thyroid-associated ophthalmopa-
thy [1,2]. Next-generation RNA sequencing and transcriptome profiling offer an opportunity to unveil molecular features of diseases [2–5],
which requires the extraction of sufficient high-quality RNA from biological samples. Although the traditional method of RNA extraction
has proved effective in normal samples like muscles or cells [6–8], obtaining sufficient high-quality RNA from adipose tissue is chal-
lenging due to high lipid content and low cell numbers in adipose tissue [9]. High-quality RNA refers to RNA that has a higher degree
of integrity and purity. RNA purity for assessing protein and salt contaminants is based on the A260/280 and A260/230 absorbance
ratios. The integrity of RNA is assessed by visualization of the 28S and 18S ribosomal RNA bands via electrophoresis analysis, and
by RNA Integrity Number (RIN), a commonly used metric for assessing the integrity of RNA samples that ranges from 10 (intact) to 1
(totally degraded) [10,11]. This study provides a step-by-step protocol yielding sufficient and qualified RNA from lipid-rich specimens for
downstream applications.

Materials & methods

Preparation of specimens & grouping
Orbital fat samples were collected from patients with thyroid-associated ophthalmopathy during the surgery and put into liquid nitrogen
immediately. Human tissue samples were obtained with patient informed consent and approval of the Institutional Review Board at
Zhongshan Ophthalmic Center.

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 p = 0.2505 p = 0.001087
2000
2.1

1500 2.0
Concentration (ng/ul)

A260/A280
1.9
1000

1.8

500
1.7

0
Classic Optimized Classic Optimized Method
Classic
Optimized

p = 0.0009994 p = 7.005e-05
2.25

2.00

6
A260/A230

1.75
RIN

5
1.50

1.25 4

Classic Optimized Classic Optimized

Figure 1. RNA yield and quality via classic and optimized methods. (A–C) Concentration and purity of extracted RNA measured by NanoDrop
Microvolume Spectrophotometer. (D) RIN of extracted RNA measured by Agilent 5400. The value of 260/230, 260/280 and RIN of RNA extracted by the
two methods were significantly different (p < 0.01).
RIN: RNA Integrity Number.

RNA extraction from orbital fat specimen via classic & optimized protocols
We optimized some of the steps of the RNA extracting protocol based on the TRIzol reagent and applied both classic and optimized
methods to extract RNA from orbital fat specimens. The classic method was performed following the TRIzol reagent protocol provided
by the manufacturer [12], which is suitable for a wide range of cell types and tissues. The optimized protocol is presented in Box 1 and
comparisons between the classic and optimized protocols are presented in Supplementary Table 1.

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Patient ID Optimized
M 12 13 14 15 16 17 18 19 20 M 21 22

28S

18S

Patient ID Classic
M 1 2 3 M 4 5 6 7 8 9 10 11 M

28S
18S

Figure 2. Electrophoresis of extracted RNA samples. (A) Electrophoresis of RNA sample in optimized group. (B) Electrophoresis of RNA samples in
classic group.

Assessment & comparison of RNA yield & RNA quality

The concentration and purity of the extracted RNA were evaluated using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher
Scientific, MA, USA). RNA purity for assessing the protein and salt contaminants was based on the A260/280 and A260/230 absorbance
ratios. The integrity of RNA was assessed by visualization of the 28S and 18S ribosomal RNA bands via electrophoresis analysis, and
by RIN via microfluidic capillary electrophoresis with an Agilent 5400 bioanalyzer (Agilent Technologies, CA, USA). Differences in RNA
yield and RNA quality in two different protocols were compared via the Wilcoxon test. Statistical analysis and data visualization in this
study was performed using R software (R Foundation for Statistical Computing, Vienna, Austria, www.r-project.org). Unless otherwise
specified, all statistical tests were two-tailed and p-value or false discovery rate <0.05 was considered statistically significant.

Results & discussion

A total of 22 human orbital fat specimens of the same quality were split into two groups referred to as classic and optimized. RNA
extraction was conducted by the traditional method and optimized method respectively, and the concentration and quality of the RNA
yielded were assessed (Table 1).
The concentrations of RNA yield in the optimized group and the control group were not significantly different (p > 0.05; Figure 1A
& Table 2). Comparing the purity of the samples, the A260/A280 and A260/A230 optical density of the RNA from the optimized group
was commonly within the ideal range (∼2.0 and 1.8∼2.2, respectively; Table 1), and the Wilcox-test showed that the RNA extracted by
the optimized method had higher purity than that of the control group (p < 0.01 for A260/A280; p < 0.001 for A260/A230; Figure 1B
& C & Table 2). The integrity of the samples was evaluated using the Agilent 5400 (Agilent, CA, USA), which gave the RIN, a parameter
reflecting RNA integrity. The RIN of the RNA extracted by the optimized method was significantly higher (p < 0.0001) than that of the
control group (Figure 1D & Table 2), indicating the higher integrity of the optimized group’s RNA.
The RNA integrity of mammals can also be inferred by the electrophoresis strips of 28s, 18s and 5s RNA. Clear strips with few smears
in the electropherogram of the optimized group’s RNA samples were observed (Figure 2A), reflecting its relatively high purity and integrity.
In contrast, the smears contained degraded RNA and blurry strips can be seen in the electropherogram of the classic group (Figure 2B),
which indicated the lower integrity and purity of the samples.
High-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is difficult. Much research has
been conducted to seek a better way to isolate RNA from adipose tissue [3,9,13–16] but has shown unsatisfactory or unstable yield, purity
and/or integrity of RNA extracted via column-based commercial kits or traditional TRIzol usage, and presents challenges in extracting

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 Box 1. Optimized method based on TRIzol for isolating RNA from adipose tissue.
Lyse samples and separate phases
1. Place approximately 150 mg of tissue in a screw containing grinding beads and add 1 ml of TRIzol reagent. While operating other
specimens, soak the screws containing both beads and tissue in liquid nitrogen to slow the degradation of RNA. The High-Speed
Low-Temperature Tissue Homogenizer (Servicebio, Wuhan, China) was used to agitate the screws at 70 Hz, -40◦ C in the current work. Each
cycle includes 90 s of homogenization and 20 s of pause to ensure the RNA would not be degraded by excessive heat. Homogenize for 5
cycles.
2. Centrifuge the screws for 5 min at 16,000 × g at 4◦ C rather than 12,000 × g at 4◦ C to sufficiently separate the lysate from tissue fragments.
3. Transfer the clear supernatant to new tubes and centrifuge again at 12,000 × g for 5 min at 4◦ C.
4. Remove the supernatant layer formed by lipids.
5. Add 200 ul chloroform to each tube, securely cap the tube and thoroughly mix by shaking.
6. Incubate for 5 min to allow complete dissociation of the nucleoproteins complex.
7. Incubate for 2–3 min.
8. Extending the centrifugation time to 15 min at 16,000 × g at 4◦ C allows for improved separation of the mixture into a lower red
phenol-chloroform layer, an interphase and a colorless upper aqueous phase.
9. To transfer the RNA-containing aqueous phase to a new tube, tilt the tube at a 45◦ angle and carefully pipette the solution, taking care not
to collect any of the interphase or organic layer.
Isolate and precipitate RNA
1. Add 0.5 ml isopropanol precooled at 4◦ C to the aqueous phase. Precooled isopropanol can retain the low temperature of the aqueous
phase, preventing the RNA from degradation.
2. Incubate for 15 min at 4◦ C. An extended 15 min ensures the RNA precipitates sufficiently.
3. Centrifuge for 15 min at 12,000 × g at 4◦ C. Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
4. Discard the supernatant with a pipettor.
Wash RNA
1. Resuspend the pellet in 1 ml of 75% ethanol per 1 ml of TRIzol reagent used for lysis.
2. Vortex the sample briefly, then centrifuge for 5 min at 12,000 × g at 4◦ C. A higher centrifugal speed of 12,000 × g precipitates the RNA
more sufficiently.
3. Discard the supernatant with a pipettor.
4. Vacuum or airdry the RNA pellet for 5–10 min. Repeat the washing process three times, to more thoroughly remove impurities.
Solubilize RNA
1. Add 20–50 μl of RNase-free water to resuspend the pellet.
2. Proceed with downstream applications or store the RNA at -70◦ C.

RNA with sufficient yield and quality from adipose samples. These studies have developed optimizing methods for isolating RNA from
adipose tissue through combinations of column-based kits and phenol-chloroform methods, or through in-house protocols [3,9,13–
16]. However, the considerable complexity of these protocols and the various kits/materials required hamper their wide use. TRIzol is
frequently used to extract RNA from tissue and cells [17]. However, the standard process is not suitable for many kinds of tissue such as
adipose tissue. In this study, we report an optimized protocol for the extraction of high-quality RNA from human orbital fat simply based
on TRIzol reagent, which is an extremely accessible ready-to-use reagent in laboratories for the isolation of nucleic acid and/or protein
from biological samples.
The delicate nature of RNA makes it susceptible to degradation during extraction, which is influenced by several factors, including
heat and the presence of RNases. The activity of RNases is also sensitive to changes in temperature, and reducing heat can decrease
the activity of RNases in RNA degrading [9,13]. Without modifications, heat itself and heat-induced enhancement of the RNases activity
accelerated the degradation of RNA during the traditional procedure. High lipids preventing the action of the lysis buffer and low cell
numbers in adipose tissue aggravate the degree and influence of RNA degradation. We tried to reduce the degradation and impurity by
storing the tubes in liquid nitrogen, improving the centrifugal speed, extending the centrifugal time and precipitate time and precooling
the reagent. Removing the fatty layer can prevent possible lipid contamination of RNA. Homogenization in a low-temperature homoge-
nizer facilitates the thorough mixing of the tissue with the reagents, ensuring complete cell lysis and RNA release and preventing RNA
degradation by inhibiting RNase activity. Additionally, the low-temperature environment provided by the homogenizer helps maintain
sufficiently low local temperatures of the samples, further preventing RNA degradation.
TRIzol reagent, the main reagent needed in this optimized protocol, is easy to access, which is a key advantage. Compared with
other methods, this optimized protocol features inexpensive and readily available reagents and relatively simpler procedures. Although
this modified protocol may require slightly more time compared with commercially available kits, it offers sufficient RNA yield and
quality. With all the modifications in this optimized protocol, large amounts of high-quality RNA samples for downstream research were
eventually gained from human orbital fat tissue. This optimized protocol might also be applied in other lipid-rich tissue directly or after
modifications, and benefits researchers interested in molecular signatures regarding lipid-rich tissue.

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Conclusion
In conclusion, the purity and integrity of RNA extracted by this optimized protocol based on TRIzol are significantly higher compared
with the traditional protocol, while there was a decreasing trend in RNA yield, which was not statistically significant. The modifications
of the method aim to achieving maximum yield by promoting complete reactions, suppressing RNase activity and creating a locally low-
temperature environment to minimize degradation and obtain RNA with higher integrity. Compared with other optimization efforts via
combinations of column-based kits and phenol-chloroform methods, or through in-house protocols, this optimized TRIzol-based method
is more effective and accessible.

Future perspective
Our understanding of adipose tissue has expanded over the past decade, with work identifying human adipose tissue as an organ with
anatomical, functional and genetic diversity. Going forward, fat will ideally be considered as relevant and influential as other organs in
maintaining human health. The challenges encountered in RNA extraction from adipose tissue can be effectively and ideally overcome in
future years. RNA extraction methods are expected to become more widespread and accessible, and research may focus on integrating
RNA extraction with downstream technologies for gene expression and functional studies.

Executive summary
• Since high-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is rather difficult, an
optimized TRIzol-based protocol that enables effective extraction of sufficient high-quality RNA from adipose tissue was developed.
Materials & methods
• A total of 22 human orbital fat specimens of the same quality were split into two groups and RNA were extracted by traditional method and
optimized method.
• The quality and quantity of RNA obtained from the two groups were measured and compared.
Results & discussion
• Based on the experimental results, the purity and integrity of the RNA extracted by the optimized protocol are significantly higher compared
with the traditional protocol.
• Compared with other column-based kits and phenol-chloroform methods, this optimized protocol features inexpensive and readily
available reagents and relatively simpler procedures.
Conclusion
• An optimized protocol for extracting sufficient and qualified RNA from lipid-rich specimens simply based on the TRIzol reagent, which is
more efficient and accessible than existing methods, was reported.

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/
suppl/10.2144/btn-2022-0120

Author contributions
R Lu supervised the project. H Zhang and Y Liu designed the study. H Zhang and B Yu conducted the experiments. Y Liu and H Zhang
performed the data analysis. H Zhang, Y Liu and R Lu drafted and edited the manuscript.

Financial & competing interests disclosure

The work was financially supported by the High-level Hospital Construction Project (303010406) and the Natural Science Foundation of
Guangdong Province, China (general program: 2023A1515012354, 2022A1515011609). The funding organization had no role in the design
or conduct of this research. The authors have no other relevant affiliations or financial involvement with any organization or entity with
a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

This research was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board at
Zhongshan Ophthalmic Center. Human tissue samples were obtained with informed consent from patients at the Zhongshan Ophthalmic
Center, Guangzhou, China.

Data sharing statement

Some or all data generated or used during the study are available from the corresponding author by request.

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Open access
This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit
http://creativecommons.org/licenses/by-nc-nd/4.0/

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