Cómo citar: Benedetti I, De León L, Reyes N. Efectos del tiempo de almacenamiento y el volumen de tejido en rendimiento
y calidad del ARN, y nivel de expresión de RNU6, de tejidos con cáncer de próstata fijados con formalina e incluidos en
parafina. Ciencia e Innovación en Salud. 2021. e128: 213-225 DOI 10.17081/innosa.128
Effects of storage time and tissue volume in the yield and quality of RNA,
and expression level of RNU6, from formalin-fixed paraffin-embedded
prostate cancer tissues
Efectos del tiempo de almacenamiento y el volumen de tejido en
rendimiento y calidad del ARN, y nivel de expresión de RNU6, de tejidos con
cáncer de próstata fijados con formalina e incluidos en parafina
Inés Benedetti
1 *
, Laura De León
2
and Niradiz Reyes
1
1
Universidad de Cartagena. Cartagena, Colombia.
2
Universidad de Los Andes. Bogotá, Colombia.
*Dirigir correspondencia a: ebenedettip1@unicartagena.edu.co
ABSTRACT
Background: Molecular analyses of tumor RNA expression have become widely used both for
research and clinical purposes. Tumoral tissue preservation is a critical step to ensure accuracy
of molecular-based diagnostics, for which, formalin-fixed and paraffin-embedded (FFPE) tissues
represent a valuable source of clinical samples. MicroRNAs are ideal biomarkers in FFPE-tissues,
in whose expression evaluation RNU6 is one of the genes used as a normalizer. Our aim was to
determine, in FFPE tissue samples, the effects of length of storage and corresponding volume of
each studied sample, on the RNA retrieval, quality and concentration, as well as their correlation
to the expression level of RNU6. Methods: Fifty tissue blocks with a mean length of tissue storage
of 30 months (SD=±12.07, 95% CI= 27.4-34.3). were included. Total RNA was isolated,
absorbance and concentrations were determined and correlated with length of storage and volume
of tissue. RT-qPCR for RNU6 was performed and their Ct results were correlated to the same
parameters. Results: There was a direct correlation between the concentration and quality of the
obtained RNA, and an inverse correlation between the tissue storage time and the RNA quality.
The volume of tissue studied was not correlated with the RNA quality or concentration. The RNA
quality and the length of tissue storage directly correlated to the RNU6 expression level, while
RNA concentration and the volume of tissue studied did not affect it. Conclusions: There is an
association between longer FFPE tissue storage time with lower RNA quality and lower RNU6
expression level.
Keywords: RNA; microRNA; nucleic acids; tissues.
RESUMEN
Introducción: Los análisis moleculares de la expresión tumoral de ARN se usan ampliamente
tanto con fines clínicos como de investigación. para ello, los tejidos fijados con formalina e
incluidos en parafina (FFPE, por su sigla en inglés) representan una valiosa fuente. Los
microARNs son biomarcadores ideales en tejidos FFPE, siendo RNU6 uno de los normalizadores
usados en la evaluación de su expresión. Nuestro objetivo fue determinar, en muestras de tejido
FFPE, los efectos del tiempo de almacenamiento y el volumen de tejido estudiado, sobre la
recuperación, calidad y concentración de ARN, en relación con el nivel de expresión de RNU6.
Métodos: se incluyeron 50 bloques de tejido con almacenamiento medio de 30 meses
(DE=±12.07, IC 95%=27.4-34.3). Se extrajo el ARN total, se determinaron absorbancia y
concentración y se correlacionaron con tiempo de almacenamiento y volumen de tejido. Se
realizó RT-qPCR para RNU6, sus resultados se correlacionaron con los mismos parámetros.
Resultados: Hubo correlación directa entre concentración y calidad del ARN, e inversa entre el
tiempo de almacenamiento del tejido y la calidad del ARN. El volumen de tejido no se correlacionó
con la calidad o concentración del ARN. La calidad del ARN y la duración del almacenamiento
de tejido se correlacionaron con el nivel de expresión de RNU6, pero no se vio afectada por la
concentración de ARN y el volumen de tejido estudiado. Conclusiones: A mayor tiempo de
almacenamiento del tejido FFPE se observa menor calidad y concentración del ARN, y menor
nivel de expresión de RNU6.
Palabras clave: ARN; microRNA; ácidos nucleicos; tejidos.
Editorial History
Receive: 19 10 2020
Accepted: 09 07 2021
Published: 21 07 2021
DOI 10.17081/innosa.128
©Copyright 2021.
Benedetti
1
et al.
214
I. INTRODUCTION
The molecular diagnosis of genetic alterations and mRNA, microRNAs or protein expression
profile of neoplasms are essential in diagnosis and treatment of cancer patients; some RNA-
based gene expression profilings are used to subclassify tumors into gene expression signatures,
and have proven to be useful prognostic and predictive biomarkers of response to anticancer
therapies; while, microRNA signatures are being investigated as diagnostic and prognostic
biomarkers in several tumor types (1). Accuracy and reproducibility of this molecular diagnosis
depends on the quantity and quality of the biomolecules obtained from tumoral tissue specimens
(2). The techniques used for tissue preservation represent critical steps to ensure a suitable, high-
quality and adequate amount of these biomolecules (3,4).
The fixation, used to preserve tissue morphology, has also focused on preserving the tissue
molecular integrity (5). The fixator used routinely is formalin at 10%, due to its low cost, fast and
complete penetration (4), and excellent conservation of morphology (5,6). The tissues are
denominated formalin-fixed and paraffin-embedded (FFPE), given the processing they go
through following fixation, that facilitates its conservation at room temperature for long periods of
time (7,8). The FFPE tissues represent a valuable source of clinical samples archived in
pathology laboratories, they are a useful tool for risk stratification, identification of prognostic
markers (8,9), and a highly valuable source of genetic material for molecular analyses both in
research and clinical diagnostics (10).
The ideal fixation method should provide a balance between the preservation of tissue
morphology, and the quantity and quality of nucleic acids obtained, especially RNA (11), but the
active ingredient in formalin, formaldehyde, leads to cross-linking between proteins and
nucleotides and causes DNA denaturation (2,12). This can lead to inconsistent results or failures
in molecular analysis, resulting from partial degradation or chemical modifications to DNA, RNA,
or proteins. Other factors that can affect the quality of nucleic acids are the tissue fixation time
and tissue exposure to oxidation, extreme temperatures or light (13). It has been suggested that
these alterations are accentuated with prolonged fixation, leading to additional degradation during
the storage period (4,5,9).
Results in relation to the RNA degradation during the FFPE tissue storage time have been
divergent and RNA obtained from archival FFPE tissue samples can vary widely both within and
across studies; previously have been reported that it is possible to extract mRNA from minute
FFPE samples but, the quality of the mRNA in these samples significantly decline with increasing
sample age (14); while others, reported no correlation between age of the FFPE tissue blocks
neither RNA yield or integrity of the extracted RNA, from archival FFPE prostate biopsies (15),
and that RNA retrieved from FFPE tissues may be successfully used for molecular analysis (16).
MicroRNAs are ideal biomarkers in FFPE-tissues because, although RNA can potentially be
degraded in them, the possibility of development of the described cross-links is greater in longer
RNA molecules; thus, they can be obtained more easily and it is possible to evaluate their
expression level from these samples (9,17). Considering this and that molecular analyses of RNA
expression using FFPE tissue have become widely used, both for research and clinical purposes,
the objective of this study was to determine, in FFPE prostatic tissue samples, the effects of
length of storage and the volume of tissue studied, on the RNA retrieval, quality and
215
concentration, as well as, on the expression level of the U6 small nuclear RNA reference gene
(RNU6), which is used as a normalizer to evaluate microRNAs expression, as an important step
in the molecular diagnosis of the neoplasms.
II. METHODS
2.1. Study design
The study was conducted at the School of Medicine, University of Cartagena, Colombia. A
group of FFPE prostate tissue blocks and their data were obtained from the Department of
Pathology at the Hospital Universitario del Caribe. The FFPE tissue blocks were manually
dissected and cut, total RNA was isolated, the quality and concentration of RNA obtained from
FFPE tissues were evaluated in relation to the length of tissue storage and the volume of each
studied sample, and, to determine the influence of these parameters in the expression level of
RNU6, quantitative real-time-PCR assays were done, and their correlations to these samples
Ct of RNU6 were calculated. The assays were conducted at the School of Medicine, University
of Cartagena. The study was approved by the local ethics committees, no patient data appear
in this article, and no experiments were performed on humans or animals for this study.
2.2 Samples selection
Fifty FFPE tissue blocks were selected from the Department of Pathology at the Hospital
Universitario del Caribe (Cartagena, Colombia). All samples were from radical prostatectomy
and trans-urethral prostatectomy specimens resected from patients with a previous diagnosis
of localized prostate cancer, who underwent surgical resection as their primary treatment. For
each patient, H&E stained slides were previously revised and specific tissue areas were
selected and dissected in the way that is described below.
2.3 Laboratory assays
Obtaining manually dissected tissue from FFPE prostate tissue
The manual dissection of the FFPE tissue blocks, to obtain specific tissue areas, was performed
in the Histopathology Laboratory at the School of Medicine, University of Cartagena. Excess of
paraffin was removed from the blocks, the area to be studied was drawn and measured in the
H&E slide and the corresponding block by a Pathologist expert in prostate cancer diagnosis;
the blocks were manually carved to cut only that area. The tissue blocks were placed in a Leica
®
microtome (Buffalo Grove, IL, USA), which blades were cleaned after cutting each block to
avoid contamination; after discarding the first three cuts, two to six cuts with a thickness
between 10 and 15 um were taken (depending on the tissue area chosen), which were directly
deposited into a 1.5 ml microcentrifuge tube previously labeled, for each of which the volume
of tissue was calculated.
RNA isolating from manually dissected FFPE prostate tissue sections
Total RNA from these samples was extracted using the miRNeasy FFPE
®
commercial kit
(Qiagen
®
Germantown, MD, USA), specially designed to purify RNA from FFPE samples. Each
216
tissue cut was incubated for fifteen minutes, at 56 ºC, in 160 μL of deparaffinization solution
®
(Qiagen
®
Germantown, MD, USA) to melt the paraffin. Then, 150 µL of lysis buffer (PKD) was
added, centrifuged for one minute at 11,000 g, the lower clear phase was removed, 10 µL of
proteinase K was added and it was first incubated for 60 minutes at 56 ºC to release the RNA
from tissue samples, and then for fifteen minutes at 80 ºC to reverse the modifications to the
RNA produced by formaldehyde. The clear lower phase was transferred to another tube and
centrifuged for fifteen minutes at 20,000 g; the supernatant was transferred to another tube and
incubated at room temperature for fifteen minutes with DNAse and DNase booster buffer to
eliminate genomic DNA. Subsequently, the samples were mixed with 320 μL of RBC buffer,
and 1120 μL of ethanol, placed in the elution column, washed twice with RPE buffer, and finally
the RNA was eluted with 20 μL of RNAse-free water.
The concentration and quality of total RNA were evaluated spectrophotometrically to determine
their absorbance at wavelengths of 260/280 using NanoDrop 2000c
®
(Thermo Scientific
®
Waltham, MA, USA).
RNU6 detection
The miScript PCR
®
kit (Qiagen
®
Germantown, MD, USA) which allows the detection of multiple
microRNAs from a single cDNA, was used to determine the expression level of RNU6.
cDNA synthesis
For each sample, was obtained cDNA from total RNA isolated from the FFPE tissues. cDNA
synthesis was performed using the miScript II RT
®
commercial kit (Qiagen
®
Germantown, MD,
USA), whereby mature microRNAs are polyadenylated by a poly-A polymerase and then
reverse transcribed into cDNA using oligo-dT primers. For this, the 5X miScript HiSpec buffer
included in the kit, specific to prepare cDNA for the subsequent quantification of mature
microRNAs was used. In detail, 2 µg of RNA were taken from each sample to prepare the
reverse transcription master mix: 4 µL of 5X miScript HiSpec Buffer, 2 µL of 10X miScript
Nucleics Mix, 2 µL of miScript Reverse Transcriptase Mix, RNA volume equivalent to 2 µg, and
variable volume of water, for a final reaction volume of 20 µL. Incubated at 37 ºC for 60 minutes,
and then incubated at 95 ºC for 5 minutes to inactivate the miScript Reverse Transcriptase Mix.
The cDNA was stored at -20 ºC until it was used in the RT-qPCR assays.
Quantitative real-time PCR
RNU6 expression levels from total RNA isolated from each sample were determined by
quantitative real-time PCR using the commercial kit miScript SYBR Green PCR
®
(Qiagen
®
,
Germantown, MD, USA), in a StepOne Real-Time PCR System® (Applied Biosystems, Beverly,
MA, USA). Each sample was analyzed in duplicate and a blank was used in each reaction.
Melting curves were acquired to monitor the quality of the reaction.
2.4 Statistical analysis
The data were registered using Microsoft Excel
®
software (Washington, USA). Differences
between the groups of tissue with different storage time an volume were determined by ANOVA
test. To study the influence of length of tissue storage and volume of tissue in the quality and
217
concentration of RNA, and also their effects in the expression levels of RNU6, were calculated
correlations using Spearman's correlation. Statistical analyses were performed using
GraphPad Prism
®
v7.00 software (Graph-Pad Software Inc, San Diego, CA); a p value <0.05
was considered statistically significant..
III. RESULTS
RNA was isolated from manually dissected FFPE tissue samples from fifty blocks with prostate
tissue, with a mean length of tissue storage of 30 months (SD= ±12.07), and a mean volume of
tissue studied of 1.38 mm3 (SD= ± 0.791), (Table 1).
The mean value of the RNA absorbance at wavelengths of 260/280 was 1.75 (± SD 0.115)
(Table I). When comparing the absorbance between the tissues with different storage times, a
significant difference was found (p = 0,0076, ANOVA test); in the post hoc test a significant
difference in absorbance ratios at 260/280 wavelengths was observed between the RNA
obtained from blocks stored for less than 18 months, and those obtained from blocks stored for
43 to 48 months (p = 0,0069, Tukey test) (Table 2).
Table 1. Characteristics of the FFPE prostate tissue blocks, purity and concentration of the
RNA obtained
Characteristic
Mean ( ± SD)
(n=50)
95% CI
Length of tissue storage
(months)
(years)
30.9 (± 12.07)
2.53 (± 1.003)
27.47 - 34.33
2.24 - 2.81
Volume of tissue studied (mm
3
)
1.38 (± 0.791)
1.15 – 1.60
Absorbance relation at 260/280 wavelenght
1.75 (± 0.115)
1,72 - 1,79
RNA concentration (ng/L)
221.8 (± 214.3)
160.9 - 282.7
Source: authors' elaboration
The RNA concentrations were between 15.5 and 1008 ng/μL, (mean 221.8 ng/μL), No
significant difference was found between the RNA concentrations from the blocks stored for
different periods of time (<18 months to 48 months), (ANOVA Test) (Table 2).
The correlation between RNA concentration and the RNA purity, determined by their
absorbance at wavelengths of 260/280, and the correlation of these parameters with length of
tissue storage, and corresponding volume of each studied sample, were determined by
Spearman's correlation. The results show a clear direct and statistically significant correlation
between the RNA concentration and the RNA quality (Rho=0.612, moderate positive
correlation, 95% CI=0.396 to 0.765, p<0.0001), (Figure 1). A statistically significant and
negative correlation between length of tissue storage and RNA purity was observed (Rho= -
0.393, low negative correlation, 95% CI=-0.611 to -0.120, p=0.048), while the RNA
concentration was not correlated to the length of tissue storage (Rho= -0.174, 95% CI= -0.438
to 0.119, p=0.228), (Figure 2).
218
Table 2. Characteristics of the RNA obtained in relation to the length of FFPE tissue
storage.
Length of FFPE prostate tissue storage (months)
p Value
18-24
months
25-30
months
31-36
months
37-42
months
(n=6)
(n=10)
(n=8)
(n=5)
RNA characteristics
p value
Post hoc test
Adjusted p
value
Relation
260/280
1,732
(0,061)
1,757
(0,103)
1,809
(0,135)
1694
(0,061)
0,0076*
(1,668-
1,796)
(1,683 –
1,831)
(1,695-
1,922)
(1,617-
1,771)
0,0069**
Concent
ration
(ng/L)
75,11
(46,25)
260,5
(208,8)
291,0
(320,3)
77,26 (44,3)
0,1918*
(26,57-
123,7)
(111,1-
409,9)
(23,15-
558,8)
(22,14-
132,4)
*ANOVA test, **Tukey test
Source: authors' elaboration
Figure 1. Association between quality and concentration of the RNA obtained from the
FFPE tissue samples
Source: authors' elaboration
219
The concentration and quality (determined by their absorbance at wavelengths of 260/280) of
the RNA obtained from the FFPE prostate tissue samples, were directly associated.
Correlation coefficients (rho) are indicated, statistical significance is represented by asterisks,
(Spearman's correlation).
Figure 2. Association between length of tissue storage, and, purity and concentration of the
RNA obtained from the FFPE tissue samples
Source: authors' elaboration
The purity of the RNA obtained from the FFPE prostate tissue samples stored for 1 to 4 years
was correlated with the length of tissue storage. The RNA absorbance at wavelengths of
260/280 ratios were negatively correlated with the length of tissue storage (left), while the
RNA concentration was not correlated to the time of tissue storage (right). Correlation
coefficients (rho) are indicated, statistical significance is represented by asterisks,
(Spearman's correlation).
The volume of tissue studied was not associated with the RNA quality (Rho= 0.273, 95% CI=
-0.163 to 0.400, p=0.054), nor the RNA concentration obtained (Rho= 0.128, 95% CI= -0.163
to 0.400, p=0.372).
RNU6 expression levels in the prostate tissue samples
To study the influence of RNA quality and concentration, volume of tissue studied and length
of tissue storage, in the expression level of RNU6, the correlations between Ct of RNU6 and
those parameters were calculated using Spearman's correlation. RNA absorbance ratio at
wavelengths of 260/280 was negatively correlated with the Ct of RNU6, indicating that RNU6
expression level could be affected by the RNA quality. Meanwhile, there was a trend towards
a negative correlation between RNA concentration and RNU6 expression level (Rho= -0.276,
220
p = 0.510). The length of tissue storage was positively correlated with the Ct of RNU6,
indicating that the longer the storage time, the lower the obtained RNU6 expression level (Fig.
3). No correlation was observed between the volume of tissue studied and the RNU6
expression level (Rho= -0.095, 95% CI= -0.371 to 0.196, p=0.510).
The correlations between the Ct of RNU6, with the concentration and quality (determined by
the absorbance at wavelengths of 260/280) of the RNA obtained from the FFPE prostatic
tissue samples, and, with the length of FFPE tissue blocks storage, were determined. The
RNA absorbance at wavelengths of 260/280 was negatively correlated with the Ct of RNU6
(top), while the RNA concentration showed a trend towards a negative correlation with the Ct
of RNU6 (middle). The length of tissue storage was negatively correlated with the Ct of RNU6
(bottom). Correlation coefficients (rho) are indicated, statistical significance is represented by
asterisks, (Spearman's correlation).
IV. DISCUSSION
The archives of FFPE tissues are a valuable resource to study genetic alterations, changes
in gene expression of mRNA, microRNAs, and proteins in tumor tissue. These samples can
be stored for a long time at room temperature without loss of integrity and offer an historic
register of tumor histology and molecular profile that could be correlated to disease evolution
(18,19). However, manipulation of human tissue specimens is known to cause changes in
their composition and structure, including nucleic acids (20), and processing of long-term
FFPE stored samples demonstrates changes in their nucleic acids content by degradation
(13,16). In this study, we observed a mild loss in the quality of the transcripts obtained from
FFPE samples, which increases with longer time of tissue storage; conversely, the RNA
concentration was not correlated to the length of tissue storage (Figure 2).
Previously, Nam et al (2), amplified shorts DNA or RNA sequences in most of their FFPE
tissue samples, they obtained the best yield in RNA quality from recently processed samples
compared to the samples stored for prolonged time. They reported that RNA integrity was less
affected during the first year, than during longer periods of storage time (2). Von Ahlfen also
describes that only after storing for more than twelve months, or at elevated temperatures, the
RNA integrity becomes a limiting factor for the PCR performance (21). Similar findings were
reported by Scorsato and Telles, who did not observe loss of material, or changes in RNA
purity related to the age of the tissue block (22).
In relation to the influence of the tissue area studied on the yield of RNA obtained, Doleshal
et al, reported that some types of tissue with a larger area on the block cut surface had higher
performance than specimens with smaller surfaces, such as skin and mammary gland (23).
In this study instead, there was not variation in the yield of RNA obtained in relation to the
volume of tissue studied, this could be related to the fact that it was tried to obtain similar
volumes of tissue, increasing the thickness of the cuts in the samples with less surface area.
Similar to the results of Carlsson in their study on prostate biopsies (15), there was not
difference in the RNA quality between samples with different volume, which rules out the need
for large amounts of tissue for adequate molecular identification.
221
Figure 3. Association between length of tissue storage, purity and concentration of the RNA
obtained from the FFPE tissue samples, with RNU6 Ct.
Source: authors' elaboration
The Johns Hopkins Pathology group demonstrated that processing to which FFPE tissue is
subjected resulted in lower performance in obtaining RNA than an equivalent amount of frozen
222
tissue. But expression profile analysis showed close to 80% agreement in the differences in
expression between recently FFPE tissues and frozen tissues. They proposed that cylinders of
FFPE tissue could be taken to store at lower temperatures, or, to store RNA obtained shortly
after tissue processing to allow molecular studies without compromising current surgical
pathology routines (24).
The alteration in amplification generated by formalin seems to correlate positively to the fixation
time and the amplicon length (25). However, despite this, biomolecules can be recovered and
analyzed using specific extraction protocols, optimized for FFPE tissues (7,26), as many of the
modifications can be reversed by heating before hybridization, and reverse transcription (25),
and the assays based on the PCR technique have been optimized to overcome the technical
difficulties generated by this type of samples (27). For example, Rodrigues Gouveia et al.,
implemented an additional wash step with PBS in DNase/RNase-free water during FFPE tissue
sample preparation for the RNA extraction, with a significant improvement in the RNA quality,
and the amplification results. They proposed that possibly washing favors the elimination of
fixative residues in tissues, which are contaminants that can act as PCR inhibitors, and also
reported that amplification was not influenced by the age of tissue block (6).
Comparative analysis of frozen and FFPE tissues suggests that microRNAs are unusually
stable and easier to retrieve from FFPE when compared to mRNA transcripts owing to their
small size (28-30). However, diverse conclusions have been reported regarding the stability of
microRNAs in FFPE tissues stored for long periods of time. Nonn et al., reported good
correlation between microRNAs expression profile in frozen tissues and FFPE tissues (17).
Siebolts et al., and Szafranska et al., found similar results on microRNAs expression in FFPE
tissues compared to frozen tissues (18,31). In contrast, Boisen et al., reported that global mean
yield of microRNA is lower in tissues with increased fixation time and in older paraffin blocks
(32). And Peskoe et al., found that tissues stored for a long time, greater than twelve years,
had a decrease in the amount of microRNA and RNU6 (12,13). In this study, like this previous
report, there was an association between longer tissue block storage time with lower RNU6
expression level.
A recent review of the literature concluded that is exceedingly difficult to harmonize the quality
criteria for human tissue samples because their great heterogeneity and the large number of
pre-analytical factors associated. They proposed to assess the integrity of tissue itself and its
derived biomolecules, to evaluate if stored human tissue samples fit for the purpose for which
they were collected (20). In this sense, although the use of FFPE samples has increased in
biomedical research to evaluate the genetic and molecular basis of diseases and the outcome,
survival and new therapies for patients, their handling, processing and storage can alter their
characteristics and influence their quality, integrity and/or molecular composition. These
aspects should be considered to take advantage of the enormous potential of these samples
for their use in precision medicine.
Author Contributions: Conceptualization, I.B. and N.R.; methodology, I.B. and N.R.;
validation, I.B. and N.R.; analisis, I.B. and N.R.; research, L.D. ; resources, I.B and L.D..; data
curation, I.B and L.D.; writing: preparation of the original draft, L.D.; writing: review and editing,
I.B. and L.D. ; visualization, L.D.; supervision, I.B.; project administration, I.B.; acquisition of
funds, I.B. All authors have read and accepted the published version of the manuscript.
223
Funding: This research was funded by the University of Cartagena, Colombia, Grant # 021-
2015 to the project: Evaluación inmunohistológica de la expresión de genes y microRNAs
asociados al cáncer de próstata y su relación con la progresión.
Acknowledgments: To the Department of Pathology at the Hospital Universitario del Caribe,
Cartagena, Colombia for their collaboration, and to the University of Cartagena for the financial
support of this research, Grant 021-2015.
Conflic of interes: The authors declare no conflicts of interest.
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