ORIGINAL ARTICLE
Optimal preservation of liver biopsy samples for downstreamtranslational applications
Alana R. Sherker • Vera Cherepanov •
Zahra Alvandi • Ryan Ramos • Jordan J. Feld
Received: 11 September 2012 / Accepted: 15 January 2013 / Published online: 12 February 2013
� Asian Pacific Association for the Study of the Liver 2013
Abstract
Objective: Molecular analysis of liver biopsy samples
from patients requires ideal tissue preservation and handling
to yield suitable material for laboratory analysis. Biopsy size,
tissue handling and preservation method all may affect the
quality and quantity of DNA, RNA and protein that can be
extracted from liver biopsy samples.
Method: In the present study, murine liver biopsies were
performed and stored under various conditions: snap-freez-
ing, RNAlater and Allprotect. Yield was compared to fresh
biopsy tissue.
Results: Fresh tissue generated the highest yield of RNA
while samples subjected to the snap-freezing generated the
lowest yield of RNA. Preservation in RNAlater yielded
higher quantities of RNA than storage in Allprotect, par-
ticularly with larger biopsy samples. There was a non-
significant trend toward improved RNA quality with
RNAlater (p = 0.35). DNA and protein yield were similar
with RNAlater and Allprotect under a number of handling
condition. Errors in tissue handling such as delays in tissue
submersion or freezing did not significantly affect tissue
yields in either preservation solution. Tissue yield was
unchanged with up to three freeze-thaw cycles in both
solutions. Biopsy size (5 vs 2 mm) and width (15 vs 18 g)
had a marked effect on tissue yield.
Conclusion: Ideally 5-mm biopsies with 15-gauge nee-
dles should be used to maximize yield. RNAlater provided
higher RNA yield with similar yields of DNA and protein
and was notably cheaper and easier to handle.
Keywords Liver biopsy preservation � RNAlater �Allprotect � RNA integrity
Introduction
Molecular analysis of liver biopsy tissue is often required for
translational research studies. Although various approaches
have been evaluated, there is no standardized method of
preserving tissue at the bedside to ensure high-quality sam-
ples make it to the laboratory. Samples left at room tem-
perature are subject to RNases, DNases and proteinases,
resulting in degradation. The traditional preservation tech-
nique employed has been the snap-freeze method. However,
this approach requires the use of liquid nitrogen at the
patient’s bedside, and samples must remain frozen during
transit in order to protect the tissue from degradation. Many
investigators have been disappointed with low-yield, poor-
quality results from snap-frozen tissue, particularly when
Electronic supplementary material The online version of thisarticle (doi:10.1007/s12072-013-9423-6) contains supplementarymaterial, which is available to authorized users.
A. R. Sherker (&) � V. Cherepanov � Z. Alvandi � R. Ramos �J. J. Feld
University Health Network, McLaughlin-Rotman Centre for
Global Health, Toronto General Research Institute, University of
Toronto, Toronto, Canada
e-mail: [email protected]
V. Cherepanov
e-mail: [email protected]
Z. Alvandi
e-mail: [email protected]
R. Ramos
e-mail: [email protected]
J. J. Feld
e-mail: [email protected]
J. J. Feld
Toronto Western Hospital Liver Centre, 399 Bathurst Street 6B
FP Rm 158, Toronto, ON M5T 2S8, Canada
123
Hepatol Int (2013) 7:758–766
DOI 10.1007/s12072-013-9423-6
RNA is required for analysis. Furthermore, snap-freezing is
quite cumbersome.
The commercial products RNAlater (Qiagen) and All-
protect (Qiagen) were developed in order to facilitate
preservation of tissue samples [1, 2]. Biopsies can be
submerged directly in either RNAlater or Allprotect and
left at room temperature for up to 1 week at 4 �C, for up to
6 months at -20 �C or at -80 �C for archival storage [1,
2]. The mechanisms by which RNAlater and Allprotect
protect the tissue from degradation are different. The All-
protect molecule has contact points, which envelop the
tissue and protect it from degradation [2]. Once the tissue is
submerged in Allprotect, a protective viscous layer engulfs
the tissue and makes it difficult to manipulate. RNAlater
contains an aqueous sulfate salt solution with a controlled
pH that precipitates RNases and other soluble proteins [1,
3]. Both solutions denature proteins and are therefore only
appropriate for use in experiments that do not require their
tertiary or quaternary structure or functionality [1, 2].
In addition to the method of preservation, other factors
can affect the yield and quality of tissue for analysis.
Ideally, tissue would be preserved immediately and remain
undisturbed until it is analyzed; however, this may not
always be possible. There may be delays in submerging
tissue in the preservation solution or transferring the pre-
served tissue to the freezer. Tissue may also be freeze–
thawed on multiple occasions if it is stored in individual
pieces rather than aliquots. How these factors affect tissue
quality and quantity and whether one preservation tech-
nique performs better under these conditions is currently
unknown. We evaluated the effects of different preserva-
tion approaches and different handling techniques on the
yield of RNA, DNA and protein from liver biopsy speci-
mens to clarify how precious patient samples should best
be preserved.
Methods and materials
Liver samples
Five mice (C57BL/6) were killed by exposure to a carbon
dioxide chamber for 5 min. The livers were excised and
samples (taken in triplicate) were collected and stored in
RNAlater or Allprotect or snap-frozen in liquid nitrogen
with subsequent nucleic acid and protein extraction. The
yield and quality of RNA, DNA and protein extraction was
compared in the preservation solutions to a fresh sample
extracted immediately after biopsy. The ability of RNA-
later and Allprotect to preserve tissue was also compared
between samples of different size and from samples
exposed to delays at various steps of tissue handling. To
assess the importance of biopsy length, samples were cut
with a scalpel and measured to be 5 9 1 or 2 9 1 mm
prior to being placed in RNAlater or Allprotect. To assess
the importance of biopsy width, samples obtained with a
Klatskin 15-gauge liver biopsy needle (Thermo) were
compared to those obtained with an 18-gauge Thermo
biopsy gun (B–D). Once the biopsy had been performed,
samples were immediately put in RNAlater or Allprotect.
In the assessment of the effects of tissue handling, 5 9 1-
mm biopsy samples were used. The effect of a delay in
submerging the tissue in preservation solution was com-
pared by leaving the liver sample out of solution for 0, 30
or 120 min. To assess the effect of a delay in freezing after
tissue submersion, liver samples were placed in RNAlater
or Allprotect immediately but were then left at room
temperature for 0, 8 or 24 h prior to freezing at -80 �C.
Finally, the effect of freeze-thaw cycles was assessed by
thawing and re-freezing samples stored in RNAlater or
Allprotect 0, 1 or 3 times prior to extraction.
Storage
Following the biopsy, samples were either stored in
RNAlater or Allprotect. For samples stored in RNAlater,
10 ll of RNAlater was used per 1 mg of liver tissue.
Samples were less that 0.5 cm thick in order to ensure
proper diffusion of RNAlater through the liver tissue.
Samples were immediately put on ice (unless otherwise
specified by the condition) and frozen at -80 �C according
to the manufacturer’s instructions. For samples stored in
Allprotect, 100 ll of buffer was used per 10 mg of liver
tissue. Samples were immediately put on ice (unless
otherwise specified) and transferred to -80 �C as specified
in the manufacturer’s instructions. Because of its extremely
viscous nature, a dispenser pump was used to aliquot
Allprotect.
DNA/RNA/protein extraction
AllPrep extraction kit was used to allow for extraction of
DNA, RNA and protein from each tissue sample following
the manufacturer’s instructions. Briefly, buffer RLT was
added to the samples, and they were then homogenized
with an electrical biovortexer. Following homogenization,
samples were spun down at full speed for 3 min. The
supernatant was collected and transferred to the AllPrep
DNA spin column. The column was stored on ice until the
DNA extraction step, when the column was washed with
buffers AW1 and AW2 and eluted with 100 ll of buffer
EB. Ethanol was added to the flow through which was
mixed and transferred to the RNAeasy spin column. The
flow-through from the RNAeasy column was stored for
protein purification while the column was washed with
buffer RW1 and RPE. RNA was eluted from the RNAeasy
Hepatol Int (2013) 7:758–766 759
123
column in 50 ll of RNase-free water. Buffer APP was
added to the flow-through kept for protein purification.
After incubation, the sample was spun down, and a large
protein pellet was washed with ethanol. The protein pellet
was partially dissolved in 8 M urea and 49 SDS with beta-
mercaptoethanol. The sample was boiled for 5 min, and the
supernatant was stored at -20 �C until it was used for
protein analysis.
RNA analysis
RNA was extracted from the liver biopsies and analyzed for
quality and quantity. Extracted RNA was treated with DNase
to remove contaminating genomic DNA. RNA concentration
(ng/ml) was measured using Nanodrop2000 Spectrophotom-
eter (Thermo Scientific). An equal volume of eluted RNA
from each sample was converted to cDNA and quantified
using real-time quantitative PCR (qPCR) for GAPDH. Mouse
GAPDH standard copies were run in triplicate and used to
calculate unknown mouse GAPDH copies. RNA quality was
assessed using the Agilent 2100 Bioanalyzer (Agilent Tech-
nologies, USA). Using a very small amount of RNA (200 pg),
samples are separated on a micro-fabricated chip electro-
phoretically and then detected using laser-induced florescence
[4]. The RNA generates peaks corresponding to the 18 and
28 s rRNA as well as a small peak corresponding to the 5 s [4].
Based on the shape of the electrophoretogram, the quality and
ratio of the peaks, an RNA Integrity Number or RIN, ranging
from 1 to 10 is assigned. The most intact RNA is assigned a
RIN of 10, while a RIN of 1 corresponds to highly degraded
RNA [5]. RIN values of at least 5 are required for downstream
applications such as microarray. RNA was also run on a 1 %
agarose gel in order to see the degree of degradation of the 28S
and 18S subunits.
DNA analysis
Extracted DNA was analyzed for quantity and quality. The
concentration of DNA (ng/ml) was measured using nano-
drop. DNA was then run on a 1 % agarose gel to assess
quality. QPCR was performed on the DNA with mouse
GAPDH primers that amplified genomic DNA: forward
primer: ACCCAGAAGACTGTGGATGG (20 nt); reverse
primer: ACACATTGGGGGTAGGAACA (20 nt).
Protein analysis
The protein pellet obtained using the AllPrep extraction
method was insoluble. However, by using 8 M urea and 49
SDS with beta-mercaptoethanol, a sufficient amount of
protein was resuspended. Total protein yield was assessed
using a Coomassie Blue gel. Standard concentrations of
BSA were run on the gel as well to estimate absolute
protein concentration. Protein was also analyzed by Wes-
tern blot for actin.
Results
RNAlater vs. AllProtect
RNA preservation
The yield of RNA and DNA from 5-mm biopsy samples
stored in RNAlater, Allprotect or snap-frozen in liquid
nitrogen was compared. A fresh biopsy sample subjected to
immediate extraction served as a measure of maximal yield.
Both RNAlater and Allprotect preserved RNA better than
snap-freezing (RNAlater 2,415 ± 1,242 vs. snap-freezing
210 ± 35 copies of GAPDH, p = 0.002; Allprotect 1,137
± 310 vs. snap-freezing 210 ± 35 copies of GAPDH,
p = 0.0095). Although RNAlater showed a higher yield
than Allprotect, the differences were not statistically sig-
nificant (p = 0.48) (Fig. 1a). Results were similar with
nanodrop, confirming higher RNA concentration with
RNAlater or AllProtect compared to snap-freezing (data not
shown). Notably, results were improved in all conditions
with DNase I treatment of the RNA prior to cDNA synthesis
to remove genomic DNA contamination. Because of the
low RNA yield with snap-freezing, only RNAlater and
Allprotect were compared for subsequent analyses.
To assess RNA quality, RNA was both visualized on a
1 % agarose gel and assessed using the Agilent 2100
Bioanalyzer. Biopsy samples had an average RIN of
6.7 ± 0.04 with Allprotect and 7.4 ± 0.6 with RNAlater
(Fig. 1b) (p = 0.35). Although the concentration of RNA
obtained from biopsy samples stored in Allprotect was
somewhat lower than that from samples stored in RNA-
later, both preservation solutions preserved RNA with
adequate integrity for downstream applications (Fig. 1b).
DNA preservation
DNA yield was evaluated by measurement of DNA con-
centration by nanodrop and DNA quantity by qPCR for
GAPDH. There were no significant differences in DNA yield
between samples stored in RNALater, AllProtect or snap-
freezing, with all preservation techniques leading to similar
yield compared to fresh tissue (Fig. 1c). Samples were run on
a 1 % agarose gel, which confirmed high-quality DNA with
all methods of preservation (data not shown).
Protein preservation
After nucleic acid extraction, biopsy samples were sub-
jected to protein extraction. The samples generated a
760 Hepatol Int (2013) 7:758–766
123
highly insoluble pellet that was partially solubilized in 8 M
urea. Total protein yield was analyzed by SDS-PAGE
followed by Coomassie Blue staining. Western blotting for
mouse actin was also performed. Protein yield was similar
with both preservation solutions but consistently slightly
higher from the samples stored in RNAlater compared to
those stored in Allprotect (Figs. 3a, b, 4b, c and 5b, c).
Effects of biopsy size
Biopsy size
Because biopsy size has a marked effect on the reliability
of histological evaluation, the majority of biopsy samples
are sent to pathology with only a small amount of tissue
saved for research. To determine the effect of biopsy length
on nucleic acid and protein yield, biopsy specimens of 5
and 2 mm length were compared. RNA yield was assessed
by RNA concentration and qPCR for GAPDH using equal
volumes of extracted RNA. With storage in RNAlater, the
RNA yield from the 5 and 2 mm samples was approxi-
mately proportional to the difference in biopsy size (2.3
fold). However, with Allprotect, the RNA yield from the
5-mm sample was very similar to that from the 2 mm
sample (1.1 fold). On direct comparison, there was 1.9-fold
more RNA recovered from 5-mm samples stored in
RNAlater than from those stored in Allprotect (Fig. 2a).
Data from nanodrop measurement of RNA concentration
paralleled the qPCR results (data not shown).
In addition to biopsy length, the biopsy width may vary
because of the size of the various needles used for liver
biopsy. Similar to the data on biopsy length, RNA yield
was proportional to biopsy width in samples stored in
RNAlater but not in those stored in Allprotect, and the
yield was higher (2.6 fold) with RNAlater on direct com-
parison of the 15-gauge biopsy samples (Fig. 2b).
RNA
Biopsy size also affected RNA quality. Biopsies of 2 mm
in length yielded RNA of lower quality (RIN 5.3 ± 0.15
for 2 mm vs. RIN 7.3 ± 0.32 for 5 mm, p = 0.014).
Biopsy width was less important than length with adequate
RIN values in both 15- and 18-gauge 5-mm biopsy speci-
mens (data not shown).
DNA
In contrast to the results with RNA, DNA yield was slightly
better with Allprotect than with RNAlater, particularly in
the smaller biopsy samples; however, the differences were
not significant. As expected, the 2-mm samples yielded less
DNA than the 5-mm samples (Fig. 2c). Similarly, the
biopsies performed with a 15-gauge needle yielded more
Fig. 1 RNA and DNA yield
with different liver preservation
techniques. Liver biopsy
samples of 5 mm of murine
liver were subjected to various
preservation techniques: fresh
tissue extraction (lined),
RNAlater (black), Allprotect
(white) and snap-freeze (grey).
Each preservation technique
was performed in triplicate.
a DNase-treated RNA was used
as a template for cDNA
synthesis. The cDNA was
subjected to qPCR for
quantification. b RNA integrity
was measured using an Agilent
2100 Bioanalyzer and assigned
an RNA Integrity Number
(RIN). High RIN values indicate
higher quality RNA. c Genomic
DNA was analyzed by qPCR
using GAPDH standard copies.
Samples were compared using
Student’s t test. **p \ 0.01
Hepatol Int (2013) 7:758–766 761
123
DNA than those performed with an 18-gauge needle
(Fig. 2d). DNA obtained from liver biopsy samples was
run on 1 % agarose gel, and trends were similar to the
results from qPCR (data not shown).
Protein
As expected, total protein yield was higher in the longer
(5 mm) and wider (15 g) biopsy samples (Fig. 3a–d).
However, biopsy width appeared to have a greater effect,
with very low protein yields from the 18-gauge samples.
Protein yield was adequate with both preservation solu-
tions, but consistently slightly higher from the samples
stored in RNAlater compared to those stored in Allprotect
(Fig. 3a–d).
Effects of tissue handling
Ideally, tissue is submerged into the preservation solution
(either RNAlater or Allprotect) immediately after the
biopsy is taken and then transferred to -80 �C for long-
term storage. To determine the effects of delays in tissue
submersion, samples were left out of solution at room
temperature for 30 or 120 min prior to submersion in either
Allprotect or RNAlater. Consistent with previous results,
RNA yield was higher from the samples stored in RNA-
later than from those stored in Allprotect, but there was
relatively little RNA degradation (Fig. 4a). Samples left
out for 30 min had similar RIN values to those submerged
immediately. With a 120-min delay in submersion, there
appeared to be some loss of RNA integrity with Allprotect
but not with RNAlater (Supplementary Fig. 1); however,
biological replicates were pooled prior to RIN analysis, and
thus these RIN measurements represent only one reading
and comparisons should be interpreted with caution. DNA
yield was unaffected by leaving the tissue at room tem-
perature for up to 2 h (data not shown). Protein yield was
not affected by 30 min at room temperature but by 2 h,
there was a notable loss in protein yield, which was slightly
more pronounced in the samples stored in RNAlater
(Fig. 4b–c).
Samples were left at room temperature for 8 or 24 h
after submersion in Allprotect or RNAlater to assess the
effect of delays in freezing after tissue submersion. There
Fig. 2 Effect of sample length, needle gauge and preservation
solution on RNA and DNA yield. Liver biopsy samples of murine
liver were subjected to either RNAlater (black) or Allprotect (white).
a Nucleic acids were extracted from murine liver samples of 5 mm
length or 2 mm length or b using needles of 15 or 18 gauge. RNA
yield was compared between samples of each size and width in
RNAlater and Allprotect. RNA yield was determined by qPCR for
GADPH using equal volumes of eluted RNA for cDNA synthesis.
c Genomic DNA yield and preservation were compared between
samples of 5 or 2 mm and d samples of 15 or 18 gauge in RNAlater or
Allprotect. DNA was quantified using qPCR for GAPDH. Each
condition was performed in triplicate. Samples were compared using
Student’s t test. *p \ 0.05 **p \ 0.01
762 Hepatol Int (2013) 7:758–766
123
was no loss in RNA yield or integrity with delays in
freezing. In fact, samples stored in Allprotect that were left
at room temperature for 24 h prior to freezing had a higher
yield than those that were put on ice immediately (Fig. 5a).
DNA yield was unaffected by delays in freezing (data not
shown). Protein yield was greater with both solutions in
samples left for 24 h prior to freezing (Fig. 5b–c).
Effects of freeze–thaw
To determine the ability of both preservation solutions to
prevent sample degradation with freeze–thawing, yields
were compared after 0, 1 or 3 freeze–thaw cycles. Both
solutions prevented sample degradation with no effects on
RNA, DNA or protein yield or quality with up to three
freeze–thaw cycles (Fig. 6a–c).
Discussion
Determination of the optimal method for storage of liver
biopsy samples is critical to ensure that maximal yield of
nucleic acids and proteins is obtained from these precious
samples. A comparison of different preservation techniques
showed that storage in RNAlater offers advantages over
other approaches, particularly for RNA preservation.
When comparing the RNA yield from biopsy samples,
extraction from fresh tissue was used as a measure of
Fig. 4 Effect of delay in tissue
submersion on RNA and protein
yield. Murine liver biopsies of
5 mm in length were left at
room temperature for 0, 30 and
120 min prior to being
submerged in Allprotect or
RNAlater. Each condition was
performed in triplicate. a RNA
yield was assessed by qPCR for
GAPDH with equal volumes of
RNA used for cDNA synthesis.
b Protein yield was analyzed by
Coomasie Blue staining and
compared to albumin standards.
c Protein yield was also
evaluated by Western blot (WB)
for actin. Samples were
compared using Student’s t test.
*p \ 0.05
Fig. 3 Effect of sample length, needle gauge and preservation
solution on protein yield and quality. Protein was extracted from
biopsies of a 5 or 2 mm length or b from needles of 15 gauge or 18
gauge. Samples were preserved in RNAlater or Allprotect. Protein
yield was analyzed by Coomasie Blue staining and compared to
albumin standards. Protein yield was also assessed by immunoblot-
ting for mouse actin with evaluation of the c biopsy length, d width
and preservation solution
Hepatol Int (2013) 7:758–766 763
123
maximal yield. Storage in RNAlater provided better RNA
yield than Allprotect, but both solutions preserved RNA
much better than snap-freezing and led to less tissue
damage. Unlike the snap-freeze method, Allprotect and
RNAlater protect the liver biopsy from RNases, DNases
and proteinases by precipitating them out of solution [1, 2].
Although RNA yield was somewhat higher with RNA-
later than Allprotect, both solutions provided RNA of
adequate quality for downstream applications. The Agilent
2100 Bioanalyzer was used to quantify RNA integrity by
assigning an RIN value based on the shape of the elec-
trophoretogram of RNA run on a micro-fabricated chip [4].
Because of the high content of connective tissue in liver
biopsy samples, there is often a significant amount of RNA
degradation and large variability in RIN values due to
damage during extraction [6]. Both RNAlater and Allpro-
tect preserved RNA with RIN values reliably above 5, the
minimum required for downstream applications like
microarray analysis [4]. RIN values above 7 are optimal,
and although there was some variability, most of the
Fig. 5 Effect of delay in
freezing on RNA and protein
yield. Murine liver biopsies of
5 mm in length were submerged
in Allprotect or RNAlater and
left at room temperature for 0, 8
and 24 h. Each condition was
performed in triplicate. a RNA
yield was assessed by qPCR for
GAPDH with equal volumes of
RNA used for cDNA synthesis.
b Protein yield was analyzed by
Coomasie blue staining and
compared to albumin standards.
c Protein yield was also
evaluated by Western blot (WB)
for actin. Samples were
compared using Student’s t test.
*p \ 0.05
Fig. 6 Effect of freeze-thaw
cycles on RNA and protein
yield. Murine liver biopsies of
5 mm length were submerged in
Allprotect or RNAlater and
properly stored at -80 �C for
long-term storage. Samples
were subjected to 0, 1 or 3
freeze-thaw cycles. Each
condition was performed in
triplicate. a RNA yield was
assessed by qPCR for GAPDH
with equal volumes of RNA
used for cDNA synthesis.
b Protein yield was analyzed by
Coomasie blue staining and
compared to albumin standards.
c Protein yield was also
evaluated by Western blot (WB)
for actin. Samples were
compared using Student’s t test.
*p \ 0.05
764 Hepatol Int (2013) 7:758–766
123
samples achieved this level of RNA integrity with preser-
vation in either solution [4].
DNA is much more stable than RNA or protein, and this
was reflected in the high yield compared to fresh tissue
with any of the preservation techniques assessed. Protein
yield was also relatively consistent with the various
methods; however, it is notable that protein subjected to
Allprotect and RNAlater is denatured and is thus only
suitable for assays that do not require the structural integ-
rity of the protein such as Western blotting [1, 2]. Analysis
that requires quaternary structure or functional protein
activity should be done using either fresh tissue extraction
or the snap-freeze method. In general, RNA yield was
higher with storage in RNAlater compared to Allprotect;
however; the differences were relatively modest and likely
not significant for downstream applications.
In addition to comparing the preservation solutions, the
effects of biopsy size and tissue handling on RNA, DNA
and protein yield were assessed. In optimal circumstances,
a large sample of tissue would be preserved with direct
adherence to the preservation protocol; however, this is not
always possible. The results confirm that biopsy size, both
length and width, are critical for optimal yields. Particu-
larly for protein analyses, size is critical, as smaller (and
thinner) samples had inadequate protein yields. Smaller
biopsy samples also yielded RNA of lower quality. Ideally
5-mm samples obtained with a 15-gauge needle should be
obtained.
Somewhat surprisingly, the differences in RNA preser-
vation between RNAlater and Allprotect were more pro-
nounced in the larger biopsy samples. With RNAlater, the
RNA yield was proportional to the biopsy size. However,
with Allprotect, the yield from 5-mm and 15-gauge sam-
ples was similar to that from the smaller 2-mm and
18-gauge samples. It is possible that Allprotect is only able
to stabilize a certain portion of RNA in the tissue or only
able to protect the surface and not penetrate the deeper
tissue. Thus, if the maximum amount of RNA is protected
from RNases in a 2-mm sample, increasing the amount of
tissue would not alter the amount of RNase-protected
RNA. RNAlater may be better able to penetrate tissue of
variable size and protect the RNA from RNases. In keeping
with the concept of a maximal threshold rather than a true
difference in protection from RNases, the RNA yield was
very similar with RNAlater or Allprotect preservation in
the smaller (2-mm or 18-g) samples. In contrast to the
differences seen with RNA, DNA preservation was similar
with both solutions and proportional to the biopsy size,
likely because of the overall stability of DNA rather than
better protection from DNases than RNases. The greater
stability of DNA is due to the use of the pyrimidine thy-
midine instead of uracil, used by RNA. Thymidine has a
methyl group at the 50 position, which protects DNA from
degradation. Uracil lacks a 50 methyl group, which reduces
its stability and makes it more susceptible to degradation,
particularly at room temperature, at which RNases function
efficiently [7].
Once the biopsy has been taken, it is important to handle
the tissue appropriately to prevent degradation. If the
preservation solution is not brought to the bedside, there
may be a delay in submerging the tissue into the solution.
To evaluate how such a delay would affect tissue yields,
tissue was left out at room temperature for 30 or 120 min
prior to submerging in RNAlater or Allprotect. Although
there was a decrease in RNA yield, the degradation was
modest and not statistically significant with up to a 2-h
delay in tissue submersion. Notably, however, although the
RNA yield was similar, there may have been some deg-
radation of RNA with a reduction in RIN in the samples
preserved in Allprotect. This may be because Allprotect is
more viscous and takes time to coat the tissue, whereas
RNAlater immediately inactivates RNAses. However, the
lack of biological replicates for this aspect of the experi-
ment markedly limits the conclusions. DNA yields were
unaffected by a delay in submersion. Protein yield, how-
ever, was notably decreased by 2 h at room temperature.
Therefore, although immediate submersion is likely opti-
mal, up to a 30-min delay seems to have minimal conse-
quence, while longer delays may lead to degradation,
particularly for protein.
After tissue submersion, samples are to be immediately
placed on ice (or at 4 �C) and then transferred to -80 �C
for long-term storage. Qiagen claims that samples will
remain stable for 7 days in Allprotect or RNAlater at
15–25 �C [1, 2]. There was no loss in RNA, DNA or
protein yield if samples were left at room temperature for
up to 24 h after submersion in either preservation solution.
In fact, there was a slight increase in RNA and protein
yield in samples left at room temperature. This is likely due
to better tissue penetration with both RNAlater and All-
protect with prolonged submersion prior to freezing, which
may then result in enhanced protection of the liver biopsy
from degradation by proteinases.
Thawing and refreezing samples can lead to a marked
reduction in tissue yields, particularly of RNA and protein.
Samples may thaw inadvertently with handling or even
with repeated opening and closing of freezers. Both
RNAlater and Allprotect are designed to protect tissue
from the damaging effects of freeze–thaw cycles. It was
reassuring to see that there was no loss in RNA, DNA or
protein with up to three freeze–thaw cycles.
In addition to the differences in tissue preservation,
there are also practical differences between Allprotect and
RNAlater. RNAlater is subjectively easier to use. The
mechanism of preservation is different between the two
substances; Allprotect is an extremely viscous solution that
Hepatol Int (2013) 7:758–766 765
123
uses contact points to protect the tissue, while RNAlater
contains an aqueous sulfate salt solution with a controlled
pH that precipitates RNases and other soluble proteins [1,
2]. Due to the viscous nature of Allprotect, it does not
freeze at -80 �C [2]. Even upon the dabbing of the sample
on paper to remove excess Allprotect, sufficient amounts of
Allprotect remain on the surface of the sample. This leaves
the biopsy sample harder to manipulate and homogenize
because of its adherence to plastic tubes. The difficulty of
handling tissue preserved in Allprotect may contribute to
the differences observed. Even if tissue is well preserved, if
subsequent extraction is more difficult, the yield may be
reduced. Additionally, Allprotect is five times the cost of
RNAlater.
In summary, both RNAlater and Allprotect offer better
tissue preservation than snap-freezing. Tissue yields of
RNA and to a lesser degree protein were superior with
RNAlater compared to Allprotect. Both solutions pre-
vented degradation after tissue submersion, even with
repeated freeze–thaw cycles. RNAlater has practical
advantages in terms of ease of use and cost. Biopsy length
and width were critical to tissue yields, particularly for
RNA and protein, and 5-mm, 15-gauge biopsies should be
the minimum standard used for translational research.
Acknowledgements The authors would like to thank Dr. Laura
Erdman for her generous gift of murine livers. We are also very
grateful to Kathleen Zhong for lending us murine qPCR primers. This
work was supported by the National Institutes of Health Hepatitis B
Clinical Research Network (NIH 5U01DK082874-02) and the
Canadian Liver Foundation.
Conflicts of Interest None.
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