Original Article |
Corresponding author: Oskan Tasinov ( oskan.tasinov@gmail.com ) © 2022 Pantelis Dimaras, Oskan Tasinov, Desislava Ivanova , Yoana Kiselova-Kaneva, Nadezhda Stefanova, Maria Tzaneva, Diana Ivanova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Dimaras P, Tasinov O, Ivanova D, Kiselova-Kaneva Y, Stefanova N, Tzaneva M, Ivanova D (2022) Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues. Folia Medica 64(4): 602-608. https://doi.org/10.3897/folmed.64.e63599
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Introduction: Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments.
Aim: The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression.
Materials and methods: Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligo dT, combination of oligo dT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed.
Results: The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis.
Conclusions: Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.
biopsy, cDNA, FFPE, qPCR, RNA probe
With the development of molecular biology techniques for research and diagnostics, there is a growing interest to use the vast archives of formalin-fixed paraffin-embedded (FFPE) tissue samples in these applications also.[
The most important issue when manipulating genetic material from FFPE is nucleic acid fragmentation and chemical modifications, especially formation of nucleoprotein complexes during formaldehyde fixation. The methods to overcome these issues are amplification of short sequences[
The limitation of oligo dT cDNA synthesis priming strategy for mRNA is the preferential transcription of the 3’ region, often lost in degraded samples, and the low number of intact transcripts may lead to inaccurate gene expression results.[
The purpose of this study was improvement of qualitative gene expression analysis using FFPE tissue samples by combining strategies aiming at: 1) increasing the RNA yield; 2) more efficient cDNA synthesis, and 3) improving the detection limit in qPCR reaction.
This retrospective study approved by the University Research Ethics Committee (P55/16.06.2016) was conducted at the Medical University of Varna in collaboration with St Marina University Hospital. Written informed consents were obtained in compliance with the Helsinki Declaration.
Tissue samples were collected during colonoscopy analysis as part of routine investigation for colorectal cancer at St Marina University Hospital, Varna, Bulgaria. Samples were immersed in 10% buffered neutral formalin solution for 24 hours before embedding them in paraffin according to a standard university hospital protocol (St Marina University Hospital, Varna, Bulgaria) and were stored at room temperature (RT).
The experimental design is presented in Fig.
Experimental design and workflow, including number of samples for each step followed in the present study.
Deparaffinisation of samples was performed using the following procedure: incubation of each sample with 1 mL xylene, followed by brief vortex and centrifugation at 14 000 rpm/2 min at RT. The procedure was repeated and after xylene removal the sample was washed with 1 mL absolute ethanol. Samples were centrifuged at 14 000 rpm/2 min at RT and ethanol was removed. The procedure was repeated. After ethanol removal, samples were left to air-dry. At this stage, samples were divided in three groups for methods A, B, and C, respectively, each group containing three samples.
Tissue samples were initially treated with 100 μL of Quickextract FFPE RNA extraction Lysis buffer (Epicentre, Illumina, USA). Samples were incubated at 56℃ for 30 min and further heated at 80℃ for 10 min. Then purification was performed using the RNA Clean and Concentrator 5 spin columns (Zymo Research, USA) according to the manufacturer’s protocol. Elution of RNA was performed with 15 μL of DNAse/RNAse free water and stored at −70℃.
Samples were treated with lysis buffer as described in method A. Immediately after the final incubation period (80℃ for 10 min), standard phenol based extraction was performed using Accuzol (Bioneer, USA) and isolated samples were transferred to Clean and Concentrator 5 (Zymo Research, USA) silica spin columns for purification and concentration. Phenol based extraction and column purification steps were performed according to the respective manufacturer’s protocols. RNA was further eluted, as described in method A.
Accuzol (Bioneer, USA) 1 mL was added directly to the deparaffinised tissue samples. The steps followed further were as described in method B.
The concentration and purity of isolated RNA was estimated spectrophotometrically (Synergy 2, Biotek).
For the removal of contaminating gDNA, a DNase reaction was performed adding 2 μL of DNase buffer and 2 μL of DNase I (1 U/μL) (Epicentre, Illumina, USA) to each sample following the manufacturer’s protocol.
For the synthesis of cDNA, 500 ng total RNA template was used. For all of the three replicates from methods A and B, three types of reverse transcription reactions were performed: 1) with oligo dT primer; 2) with oligo dT and random hexamer primer; 3) with gene specific reverse primer (RPL37A: Forward 5’ ATTGAAATCAGCCAGCACGC 3’ and Reverse 5’ AGGAACCACAGTGCCAGATCC 3’). Samples were transcribed using RevertAid cDNA synthesis kit (Thermo Scientific, USA) according to the manufacturer’s protocol.
Preamplification was performed in 50 μL volume PCR reaction for each sample containing: 5 μL template cDNA; 5 μL of Taq DNA polymerase (2U) (New England Biolabs, USA) buffer containing MgCl2; 2 μL dNTPs (2.5 mM); forward and reverse gene specific primers (RPL37A, see cDNA synthesis) (Sigma-Aldrich, Germany) to a final concentration of 50 nM each; and PCR grade water (Sigma-Aldrich, Germany) up to 50 μL. Samples were amplified in a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems, USA). Initial denaturation was performed at 95℃ for 5 min, followed by 95℃ for 15 s and 60℃ for 4 min for 5 cycles. Samples were finally cooled down to 4℃ and stored at −20℃. The samples were placed on ice during all preparations. Each DNA-se treated sample provided a single preampified sample.
Results were validated by qPCR using standard SYBR Green qPCR Master Mix (Thermo Scientific, USA). Reactions in total volume of 10 μL were performed for each sample (preamplified and non-preamplified) as follows: 5 μL Master Mix with ROX dye; gene specific primers (RPL37A, see cDNA synthesis) (Sigma-Aldrich, Germany) to a final concentration of 0.25 μM each and 4 μL 10× diluted preamplified or non-preamplified cDNA. Reaction conditions were as follows: initial denaturation at 95℃ for 10 min, followed by 95℃ for 15 s and 63℃ for 1 min for 40 cycles. Melting curve was added at the end of each qPCR analysis. Reactions were performed in triplicate.
Absolute quantification method was performed and standard curve was created to assess preamplification efficacy of gene specific primed cDNA strategy for RNA isolation methods A and B. A serial decimal dilutions of the non-preamplified cDNA (25 ng/μL, 2.5 ng/μL, 0.25 ng/μL, 0.025 ng/μL, 0.0025 ng/μL) were used as standard. The change in Ct value resulting from preamplification was analysed by calculating the initial concentration of the template before preamplification (C=2.5 ng/μL) and running qPCR with the same template volume of preamplified product, using SYBR Green qPCR Master Mix (Thermo Scientific, USA) according manufacturer’s protocol. RPL37A gene primer set (see cDNA synthesis) was used in performing of qPCR.
Data were analysed using GraphPad Prism V6 software. For the estimation of statistical significance, single-way and two-way ANOVA statistical analyses were performed. P values <0.05 were considered as statistically significant.
Analysis of concentration of RNAs obtained by the three different methods showed that combination of lysis buffer extraction with phenol-chloroform extraction (method B) outperformed the other methods (A and C) (Table
Comparison of RNA quantity and purity isolated using three different extraction methods
Method | RNA Concentration [ng/μL] | RNA yield [ng] | A 260/280 nm |
A | 163.36±17.96 *** | 2450.40±269.43 | 2.03±0.07 |
B | 259.87±38.60 ***, # | 3898.00±579.02 | 2.02±0.02 |
C | 15.51±2.06 | 232.60±30.90 | 1.83±0.21 |
Average Ct values of qPCR reactions of preamplified and non-preamplified samples, obtained after RNA extraction with methods A and B are presented in Table
Comparison of Ct values obtained through qPCR of non-preamplified and preamplified cDNA samples obtained after RNA extraction with methods A and B
Non-preamplified | ||
Priming strategy | Ct A | Ct B |
Oligo dT primer | 27.93±0.69** | 31.50±0.46 |
Oligo dT+random hexamer primer | 26.40±0.62## | 27.26±0.60### |
gene specific primer | 20.20±0.53###, $$$ | 20.89±0.46###, $$$ |
Preamplified | ||
Priming strategy | Ct A | Ct B |
Oligo dT primer | 26.14±0.39** | 28.54±0.49 |
Oligo dT+random hexamer primer | 18.89±2.09### | 19.29±1.49### |
gene specific primer | 17.17±0.62### | 16.99±0.46###, $ |
Gene specific primed cDNA synthesis gave significantly lower Ct values (p<0.001) for both RNA extraction methods (A and B) compared to oligo dT and to combination of oligo dT and random hexamer primers, for both non-preamplified and preamplified cDNA samples (Table
Application of oligo dT priming strategy resulted in a statistically significant lower Ct value in RNA extraction method A than in method B, on non-preamplified (p<0.01) and on preamplified cDNA templates (p<0.01). This could be attributed to the ability of lysis buffer extraction, solely, to result in higher ratio of intact to fragmented, short RNAs, compared to concomitant phenol-based extraction.[
Comparing Ct values between non-preamplified and preamplified cDNA templates for all three priming strategies to assess the efficacy of preamplification, we noticed statistically significant improvement for both RNA extraction methods A (p<0.01) and B (p<0.001) (Fig.
Comparison of Ct values obtained through two-step qPCR of non-preamplified and preamplified cDNA samples. Data are presented as mean ±SD of FFPE tissue samples for both RNA extraction methods utilizing three different reverse transcription priming strategies (oligo dT; oligo dT+random hexamer; gene specific reverse primer). **p<0.01 vs. RNA extraction method A; #p<0.05, ##p<0.01, ###p<0.001 vs. respective priming strategy and RNA extraction method w/o preamplification. A. Lysis buffer RNA extraction (grey); B. lysis buffer and phenol-based RNA extraction (dark grey).
By performing an absolute quantification, to assess preamplification efficacy of gene specific primed cDNA strategy for RNA isolation methods A (Fig.
Relative quantification for construction of standard curve of gene specific primed cDNA for calculation of the preamplification efficiency, by taking r2 (R square) value. Used standard dilutions of non-preamplified cDNA were 25 ng/μL, 2.5 ng/μL, 0.25 ng/μL, 0.025 ng/μL, 0.0025 ng/μL. Preamplified unknown cDNA sample volume was corresponding to the volume of 2.5 ng/μL standard dilution. A) RNA extraction method A; B) RNA extraction method B. Legend: standard dilutions (●); unknown sample (♦).
The commonly used formalin fixation of tissue samples limits most molecular techniques by causing nucleic acid degradation[
Three different RNA extraction methods were compared implementing basic techniques, involving a tissue lysis buffer and a phenol-based extraction, as well as a combination of both. By replacing traditional ethanol precipitation with the more advantageous silica based spin columns, we managed to provide maximum efficiency and purity of the extracted RNA. The combination produced the highest yield and purity of RNA, as expected. The phenol-based extraction failed to achieve high yield to an acceptable level and was excluded from further analysis.
As mentioned before, RNA degradation and modification compromise the reverse transcription reaction and directly affects the produced cDNA. Different priming strategies were compared including modified protocols using oligo dT, combination of oligo dT and random hexamer primers, and gene specific primed cDNA for a set of genes. The gene specific primed strategy outperformed the rest priming strategies for both RNA extraction techniques (Table
To increase the sensitivity of the analysis, we evaluated the effect of targeted preamplification of cDNAs. By comparing Ct values obtained through qPCR on preamplified and non-preamplified templates of all cDNA primed strategies for both extraction methods (A and B), we observed that preamplification can be safely used on gene specific primed cDNA templates, considering the preamplification conditions. More specifically, the 5-μl cDNA volume corresponding to 125 ng of transcribed total RNA along with a low number of amplification cycles (n=5) was chosen to avoid fluctuations due to Poisson noise. Low primer final concentration, 50 nM, and annealing-extension temperature of 60℃ decreased nonspecific amplification. To compensate for the low primer concentration, annealing time was increased to ≥3 min.[
Quantification of the preamplified template by standard curve verified the consistency of preamplification efficiency, evaluating and correcting any variations. A higher Ct value of 5.5 was estimated, compared to the 5 run cycles. Confirmation through standard curve for the consistency of change in Ct values is highly recommended.
In conclusion, the combination of lysis buffer with phenol-based extraction giving the highest RNA yield, along with gene specific primed cDNA synthesis, is of great superiority. Increasing the target gene template by preamplification decreases Ct value and achieves higher accuracy of the results.
This study was supported financially by the Medical University of Varna, fund “Science”, project P16020 .