A M-MLV reverse transcriptase with reduced RNaseH activity allows greater sensitivity of gene expression detection in formalin ﬁ xed and paraf ﬁ n embedded prostate cancer samples☆

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A M-MLV reverse transcriptase with reduced RNaseH activity allows greater sensitivity of gene expression detection in formalin ﬁ xed and paraf ﬁ n embedded prostate cancer samples☆

Rachel M. Hagen

^a,

⁎ , Anthony Rhodes

^a,1

, Jon Oxley

^b

, Michael R. Ladomery

^a,1

aFaculty of Health and Life Sciences, University of the West of England, Bristol BS16 1QY, UK

bNorth Bristol NHS Trust, Bristol, UK

a b s t r a c t a r t i c l e i n f o

Article history:

Received 27 May 2013 Available online 2 June 2013 Keywords:

FFPE

Reverse transcriptase M-MLV

PCR

Formalinfixed and paraffin embedded (FFPE) human tissue collections are an invaluable resource for retrospective gene expression studies. However formalinfixation results in chemical modification of RNA and increased RNA degradation. This can affect RNA yield and quality. A critical step when analysing gene expression is the conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase (RT) enzyme. FFPE derived RNA may affect the performance and efficiency of the RT enzyme and cDNA synthesis.

We directly compared three commonly used FFPE RNA isolation methods and measured RNA yield, purity and integrity. We also assessed the effectiveness of three commercially available Moloney Murine Leukemia Virus (M-MLV) RTs on cDNA synthesis and gene expression sensitivity when using FFPE RNA as a template.

Our results show that gene detection sensitivity is dependent on the isolation method, RT and length of the PCR amplicon (b200 bp) when using FFPE RNA. The use of an M-MLV RT enzyme with reduced RNaseH activity gave signiﬁcantly increased qRT-PCR sensitivity when using FFPE RNA derived from prostate tissue.

The choice of RT can also affect perceived changes in target gene expression and thus the same RT should be used when attempting to reproduce results from different studies. This study highlights the need to optimise and evaluate RNA isolation methods and RTs when using FFPE RNA as a template in order to maximise a successful outcome in PCR applications.

Introduction

Analysis of gene expression patterns of tumour samples can give invaluable information in studies evaluating new prognostic biomarkers and therapeutic targets (Huang et al., 2010). Formalinfixed paraffin embedded (FFPE) tissues represent a substantial archive allowing large retrospective studies to be conducted. Archival FFPE samples are even more important when studying diseases that are slow onset/growing or rare and where detailed and long term clinical follow up data is needed in order to fully assess and interpret any gene expression results. As such it is fundamental to develop and re- fine protocols that use RNA from FFPE tissues.

A number of studies have utilised RNA derived from FFPE samples to study gene expression using quantitative real-time PCR (qRT-PCR)

(Dunn et al., 2009; Lewis et al., 2001; Madabusi et al., 2006; Reis et al., 2011; Stanta and Schneider, 1991) although it still can prove problematic. RNA isolation and quality are compromised in FFPE samples with RNA often being substantially degraded. Three major phases that can affect RNA degradation and the output quality of the samples are i) delay infixation (time between tissue acquisition andfixation), ii) processing of the sample (fixation and embedding) and iii) storage (time and temperature) which retrospectively are difficult for the end researcher to control (Chung et al., 2008; von Ahlfen et al., 2007). For- malinfixation results in the alteration of RNA with the addition of mono-methylol to nucleotide bases which are followed by the electro- philic attack of N-methylol on the formation of methylene bridges between two amino groups (Masuda et al., 1999). These chemical mod- ifications have been shown to alter RNA quality and quantity isolated from FFPE samples making it challenging to conduct accurate and reliable gene expression profiling using FFPE samples (Srinivasan et al., 2002).

A popular way to assess gene expression is through the utilisation of reverse transcriptase PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) methods. The success of these methods largely depends on the quality and integrity of RNA and the successful conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase (RT) Abbreviations: CDH1, E-Cadherin;CDH2, N-Cadherin; FFPE, Formalinﬁxed parafﬁn

embedded; MMLV, Moloney Murine Leukemia Virus; qRT-PCR, quantitative real-time PCR; RT, reverse transcriptase; RT-PCR, reverse transcriptase PCR.

☆ Source of Funding: RMH is supported by the Bristol Urological Institute and the Univer- sity of the West of England.

⁎ Corresponding author. Fax: +44 1173 282810.

E-mail address:[email protected](R.M. Hagen).

1Joint senior authors.

http://dx.doi.org/10.1016/j.yexmp.2013.05.008

Contents lists available atSciVerse ScienceDirect

Experimental and Molecular Pathology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y e x m p

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enzyme. As FFPE RNA is often degraded the cDNA synthesis step needs to be as analytically sensitive, efﬁcient and reproducible as possible. One of the most popular commercially available RT is Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV). Manipula- tions of M-MLV have resulted in variants with different properties such as reduced RNAse H activity (M-MLV/H⁻) and improved thermostability (M-MLV/TS).

There are a number of protocols and commercially available isolation kits dedicated to the isolation of RNA from FFPE material (Boeckx et al., 2011; Deben et al., 2013; Okello et al., 2010). Whilst others have shown that RNA can be isolated from FFPE and used successfully in PCR, few studies have compared RNA isolation and RTs in combina- tion to assess the effect on cDNA yield and thus gene expression sensitivity in FFPE samples. In this study we focus on utilisation of FFPE RNA from prostate carcinomas. FFPE prostate samples can be particu- larly problematic as to get deep formalin penetration of the tissue prostates has to be maintained in fixation solution for over 24 h, which can reduce the quality of RNA that can be isolated (Ruijter et al., 1997). Therefore the aim of this study was to compare RNA isolation methods on RNA yield, purity and degradation using FFPE prostate carcinomas that had been stored for a range of years (3–12 years). We directly compared a commercially available spin columnfilter purification method (Qiagen FFPE RNeasy kit), a glass fibre capture spin columns (Roche High Pure FFPE RNA Micro kit) and a guanidine thiocyanate–phenol–chloroform extraction protocol (Tri Reagent). We also assessed the yield and sensitivity of commercially available wild-type and modified M-MLV RTs on gene expression by use of RT-PCR and qRT-PCR using RNA isolated by the different methods.

Materials and methods Human tissue

This study was performed under approval of the National Re- search Ethics Committee (NRES No. 09/H0102/48). The study used tissue samples from patients diagnosed with adenocarcinoma of the prostate between 2000 and 2009. Samples wereﬁxed in neutral buff- ered saline for at least 24 h before processing and the wax blocks were stored at room temperature. Three consecutive 10μm wax curls were cut from each sample case.

RNA isolation

A wax curl from six patient cases was used for RNA isolation using either A) FFPE RNeasy kit (Qiagen), B) High Pure FFPE RNA Micro kit (Roche) or C) Tri Reagent (Sigma). RNA isolation was carried out per manufacturer's recommendations with the following amendments.

Each wax curl was placed in a 1.5 ml eppendorf and deparafﬁnised using 1 ml of Histoclear at 56 °C for 5 min. Tissue was pelleted by centrifugation and Histoclear (National Diagnostics) was removed followed by two ethanol washes. The pellet was air-dried before adding lysis buffer (supplied by kits) containing proteinase K and incubated at 56 °C for 16 h. For the Tri Reagent protocol the lysis buffer (10 mM Tris–HCl, 2 mM EDTA, 0.5% Triton X-100, pH 8.0) was used.

After proteinase K digestion the sample was incubated at 80 °C for 15 min before being placed on ice for 3 min. The sample was then centrifuged and the supernatant was added to 1 ml of Tri Reagent and the standard RNA isolation protocol was followed per manufacturer's instructions. The RNA pellet was air-dried at 37 °C for 15 min to ensure no carry-over of ethanol before resuspension in nuclease free water. For each isolation protocol used the RNA was eluted in 20μl volume (nuclease free water for the Qiagen and Tri Reagent protocol; provided elution buffer for the Roche protocol). Due to lim- ited material the isolation method was performed twice for each

sample and the average RNA concentration is presented. The RNA was then pooled and used in the various reverse transcription reactions.

RNA quantiﬁcation and integrity analysis

RNA was quantiﬁed using a Nanodrop spectrophotometer (Ther- mo Fischer scientiﬁc, Delaware USA). RNA degradation and RNA integrity number (RIN) were assessed by capillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA).

cDNA synthesis

For all samples 500 ng of total RNA was reverse-transcribed using three different reverse transcriptases A: Maxima H Minus Reverse Transcriptase, M-MLV/H⁻(Thermo Scientiﬁc), B: Tetro Reverse Tran- scriptase, M-MLV/TS (Bioline) and C: Wild-type M-MLV reverse transcriptase, M-MLV (Promega) per manufacturer recommendations. A mix of Oligo(dT) and random primers was used as the priming agent. Samples were incubated at temperatures and times stated in the manufacturer's protocols (M-MLV/H⁻and M-MLV/TS: 30 min at 50 °C; M-MLV: 60 min at 37 °C). Reactions were stopped by heating at 70 °C for 15 min.

Reverse transcriptase PCR

Primers were designed to span at least one exon boundary using the Primer Express 2.0 software (Applied Biosystems, Warrington, UK) and were purchased from Sigma-Genosys (Haverhill, UK). To evaluate the expression ofβ-actin (Accession numberNM_00101.3), an upstream 5′forward primer (5′-CCTGGCACCCAGCACAAT-3′) and a 3′ reverse primer (5′-GCCGATCCACACGGAGTACT-3′) was used and gave a PCR product 70 bp in length. To evaluate the expression of 18S rRNA (Accession numberNR_003286.2), an upstream 5′forward primer (5′-ACCCGTTGAACCCCATTCGTGA-3′) and a 3′reverse primer (5′-GCCTCACTAAACCATCCAATCGG-3′) were used and gave a PCR product 159 bp in length. To evaluate the expression of GAPDH (Accession number NM_002046), an upstream 5′forward primer (5′-ACGGATTTGGTCGTATTG-3′) and a 3′ reverse primer (5′-CTCCTGGAAGATGGTGAT-3′) were used and gave a PCR product 212 bp in length. PCR was carried out using GoTaq Hot Start Poly- merase (Promega) using manufacturer's recommendations and 50 ng cDNA (thermal cycling conditions: 95 °C for 2 min, followed by 37 cycles of 95 °C for 45 s, 55 °C for 45 s and 72 °C for 45 s, and aﬁnal extension step of 72 °C for 5 min). The PCR product was examined by electrophoresis using 2% w/v agarose gel electrophoresis with 10μg/ml ethidium bromide. A DNA ladder (Hyperladder II, Bioline) was run alongside the samples to assess PCR product length.

The agarose gel was then analysed using an UV illuminator (Minibis, DNR Bio-Imaging Systems). Experiments were repeated three times.

Quantitative real-time PCR

Quantitative PCR was performed using 2 × SensiFAST SYBR Hi-ROX master mix (Bioline) using primers as described above at 300 nmol concentration on the StepOne Plus Real-Time PCR Sys- tem (Applied Biosystems). To evaluate the expression of E- Cadherin (Accession number NM_004360.3), an upstream 5′ forward primer (5′-ATTCTGATTCTGCTGCTCTTG-3′) and a 3′ reverse primer (5′-AGTAGTCATAGTCCTGGTCTT-3′) were used. N-Cadherin (NM_001792.2) expression was evaluated using an upstream 5′forward primer (5′-CTCCTATGAGTGGAACAGGAACG-3′) and a 3′ reverse primer (5′-TTGGATCAATGTCATAATCAAGTGCTGTA-3′). PCR reaction conditions were 20 s at 95 °C then 40 cycles of 3 s at 95 °C and 30 s at 60 °C. In addition a melting curve analysis was performed to check for multiple products and primer-dimer

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ampliﬁcation. Melting curve analyses were conducted by a stepwise decrease in temperature from 95 °C to 60 °C over a 35 minute peri- od with measurement of total SYBR Greenﬂuorescence every 1 °C.

The threshold cycle (Ct) which is the intersection between each ﬂuorescence curve and a threshold line, was automatically calculated by the StepOne Plus Real-Time PCR System (Applied Biosystems) using default parameters. All samples were run in triplicate on the plate and used the same conditions to allow direct comparison of threshold cycle (Ct) values. A non-template control and water only controls were run for each RT enzyme to assess for any contamination. No contamination issues were detected.

Statistical analysis

Differences in RNA yield and gene expression between groups were assessed by one-way analysis of variance (ANOVA).

Results

Quality and quantity of total RNA using different isolation methods

RNA was isolated using wax curls collected from a number of FFPE prostate blocks that had been stored for varying amounts of time (ranging from 3 to 12 years). We found that we had to amend the proteinase K digestion as the recommendation by Qiagen (15 min digestion) and Roche (3 h digestion) was not sufficient to isolate usable quantities of RNA (data not shown). We found that a longer overnight proteinase K digestion (16 h) was needed in order to isolate a sufficient yield of RNA for cDNA synthesis and this digestion time was thus used for all isolation methods. All isolation methods allowed the isolation of RNA from FFPE samples but with varying degrees with regard to yield and purity (Fig. 1). The Qiagen FFPE RNeasy kit gave consistently the greater yield of RNA with good yields of RNA from all samples. Tri Reagent also resulted in the isolation of an ade- quate amount of RNA fromfive out of six FFPE tissues. The Roche isolation method gave the least consistent results with only two out of six samples giving a yield greater than 100 ng/μl.

The purity of the RNA isolation was assessed by measuring the ratio of absorbance of 260 nm/280 nm (A260/280) and 260 nm/

230 nm (A260/230). The Qiagen kit and Tri Reagent consistently gave A260/280 ratios close to 2.0 whereas the Roche isolation method gave more varied A260/280 ratios with three out of six samples having an A260/280 ratio of 1.90 or less. The Qiagen and Tri Reagent methods gave good A260/230 values (mean 2.24 and 2.18 respectively) for

samples tested. However the Roche kit gave signiﬁcantly low A260/230 values (0.34).

To assess RNA degradation RNA from each isolation method from samples two tofive were sent for electrophoretic analysis on an Agilent Bioanalyser (Fig. 2). As expected RNA from our FFPE tissue was significantly degraded independent of the isolation method. For each sample a RNA integrity number (RIN) was calculated. There was no significant difference in average RIN values between isolation methods (Qiagen, 2.400; Roche, 2.375; Tri Reagent, 2.400).

Qualitative gene expression proﬁles using different reverse transcriptases as assessed by reverse transcriptase PCR

We next assessed the sensitivity of commercially available RTs on the RNA isolation from our FFPE prostate tissues using the RNA isolated using the Qiagen FFPE RNeasy kit and Tri Reagent. The same starting material was used for all RT reactions to allow direct comparison. RNA was converted to cDNA using three commercially available M-MLV RT enzymes: A) M-MLV with reduced RNaseH activity (M-MLV/H⁻), B) M-MLV with increased thermostability (M-MLV/

TS) and C) wild-type M-MLV (M-MLV). Reverse transcriptase PCR (RT-PCR) was performed using primer sets that produced different PCR fragment sizes to allow RNA fragmentation size and sensitivity of each reverse transcriptase (Fig. 3).

Using RNA isolated from Qiagen's FFPE RNeasy kit all RT enzymes successfully ampliﬁed all samples using primers that generated products up to 212 bp in length. The wild-type M-MLV enzyme gave a stronger signal when amplifyingβ-actin with a PCR product length of 70 bp, followed by M-MLV/H⁻and then M-MLV/TS. As PCR product length increased variation between the RTs was reduced. At- tempts to amplify longer PCR products (greater than 300 bp) did not produce any bands (data not shown).

Using RNA isolated using Tri Reagent all RTs were able to amplify fragments up to 159 bp. However when using primers that produced 212 bp fragments, greater variability was seen and a weaker signal was produced compared to those that had been generated using RNA from the Qiagen FFPE RNeasy kit under the same conditions. The wild-type M-MLV produced PCR products 212 bp in 0

2000 4000 6000 8000 10000 12000

RNeasy Kit High Pure kit Tri Reagent

RNA (ng)

**

Fig. 1.Average RNA concentration (ng/ml) isolated from FFPE prostate carcinomas (n = 6) using either the commercially available Qiagen kit (FFPE RNeasy kit), Roche kit (High Pure FFPE RNA Micro kit) or Tri Reagent (Sigma). Error bars represent standard error of the mean. ** = p-valueb0.01.

2 3 4 5

Q R T Q R T Q R T Q R T Sample ID

Ladder

4000 bp

2000 bp 1000 bp 500 bp 200 bp 25 bp

Year

2004 2006 2002 2007

Isolation Method

Fig. 2.Electrophoresis gel image of FFPE RNA samples using the 2100 Agilent Bioanalyser. RNA isolated using the Qiagen FFPE RNeasy kit (Q), Roche High Pure FFPE RNA Micro kit (R) and Tri Reagent (T) is shown.

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length in all samples. M-MLV/H⁻and M-MLV/TS enzymes appeared to give stronger signals when attempting to produce 212 bp products but PCR ampliﬁcation success was less consistent across the sample set.

Quantitative gene expression proﬁles using different reverse transcriptases as assessed by real-time PCR

We next assessed the sensitivity of the different reverse transcriptases with RNA isolated from the Qiagen FFPE RNeasy kit with regard to the detection of target gene expression using SYBR Green quantitative real-time PCR (qRT-PCR). Initially an ampliﬁca- tion curve was generated using a serial dilution of a pool of cDNA from all RT reactions to determine the linear relationship between dilution points and the suitability of the reaction to accurately determine gene expression. To assess this we used the three housekeeping genes as beforeβ-actin,18S rRNAandGAPDH (Fig. 4). In general a low Ct value corresponds to high gene expression in the biological samples. As we used the same starting material for all RT strategies a low Ct value indicates a more efﬁcient RT reaction.

The mean Ct for all housekeeping genes/RTs is shown inFig. 4. An R²value gives a good indication of how well experimental dataﬁts the regression line. For qRT-PCR experiments to be valid a R²value should be greater than 0.990. R² values of 0.99 were obtained when using primer sets that gave 70 bp and 159 bp products in length suggesting a clear linear relationship. However the linear relationship was decreased to 0.90 when a larger amplicon length (212 bp) was used. The M-MLV/H⁻enzyme gave reduced Ct values

for all genes tested compared with M-MLV/TS and M-MLV suggesting greater sensitivity and cDNA yield.

In order to further evaluate sensitivity we looked at the ability of each RT to detect E- and N-Cadherin (CDH1andCDH2respectively) mRNA expression proﬁles using a set of benign prostate (n = 6) and prostate carcinoma (n = 6) prostate FFPE samples (Fig. 5). No signiﬁcant difference inβ-actin mRNA expression was observed between benign and cancer cases and this was used as a normalisation gene. M-MLV/H⁻gave the greatest fold change in bothCDH1(+709) andCDH2(+1559) in cancer samples compared with benign. Both M-MLV/TS and M-MLV generated cDNA resulted in increased fold changes inCDH1mRNA (+85 and +8 respectively) expression in cancer samples compared with benign, however using MMLV/TS there was no detection ofCDH2using qRT-PCR. A signal was observed in samples with cDNA generated from MMLV and there was a 9-fold increase in CDH2observed in cancer samples compared with benign. The ratio of E-/N-Cadherin was reduced in cancer samples compared with benign samples using the M-MLV enzyme. In contrast no difference in the ratio between these two genes was observed between cancer and benign samples using the MMLV/H enzyme.

Discussion

In prostate cancer and other diseases long term follow up data is crucial as prostate cancer can be slow growing with the time to develop advanced disease and recurrence time varying greatly between patients. Due to the frequent occurrence of multiple tumour foci in the prostate of patients with prostate cancer, pathologists may be reluctant to risk compromising the accuracy of the diagnosis by the

18S (159bp)

-Actin (70bp)

GAPDH (212bp)

M-MLV/H

^-

M-MLV/TS M-MLV

M-MLV/H

^-

M-MLV/TS M-MLV

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

GAPDH (212bp) (70bp)

18S (159bp)

2 3 4 5 6 2 3 4 5 6 2 3 4 5 6

A) RNA Isolation – Qiagen FFPE Kit

B) RNA Isolation – Tri Reagent

β

-Actin β

Fig. 3.RT-PCR analysis using gene primer sets (β-actin, 18S rRNA and GAPDH) to produce different fragment sizes is shown using FFPE RNA from the Qiagen kit (Panel A) and Tri Reagent (Panel B). During the cDNA conversion step either MMLV with reduced RNaseH activity (MMLV/H⁻), thermostable MMLV (M-MLV/TS) or wild-type MMLV (MMLV) was used. Six Qiagen FFPE RNA samples were used. Five Tri Reagent FFPE RNA samples (2–6 from Table 1) were used. As a negative control RNA with no RT enzyme was also generated and used in the RT-PCR reactions (labelled RT neg.).

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removal of separate tissue samples for freezing and molecular studies.

Using fresh frozen prostate samples can also result in missed tumours as it is difﬁcult to see the tumours when cutting fresh prostate tissue.

In a recent paper byWarren et al. (2013) 36/150, tumours were missed using a systematic sampling method on fresh tissue. Conse- quently FFPE samples are frequently the only clinical samples available for retrospective studies. As such, archival tissue, such as FFPE samples associated with clinical data, are invaluable in order to generate meaningful conclusions based on transcriptional analysis.

Using FFPE prostate carcinomas we show that the RNA isolation method and RT conversion strategy can affect the accuracy and sensitivity of PCR applications.

The modification of RNA by formalinfixation can result in a poor template for reverse transcription and cDNA synthesis which is needed in commonly used gene quantification techniques such as qRT-PCR (Masuda et al., 1999). Therefore it is important to optimise and use RNA isolation protocols that reverse as much of the formalinfixation as possible. All isolation protocols were able to isolate RNA independent of the storage time of the FFPE tissue. In addition we found that storage length of the FFPE tissue did not adversely affect the amount or usability of RNA extracted. The Qiagen kit and Tri Reagent kit gave consistently the greatest RNA yield and purity compared with the Roche kit. Whilst Tri Reagent has not been specifically marketed towards isolating RNA from FFPE material it is an important comparison that to make as many labs will have access to this reagent

and it must be noted that it is more cost effective that the commercially available kits designed speciﬁcally for FFPE material and in our hands actually performed as well or better than the specialised kits. The decreased RNA yield observed with the Roche might be as a result of the reducedﬁlter area present in the Roche columns. As a result we found that despite only using one 10μΜ section the Roche columns did become clogged and thus smaller sections or multiple columns may be needed in order to use this kit effectively.

The Qiagen kit also recommends an incubation step at 80 °C after proteinase K digestion to facilitate reversal of the formalin crosslinking aiding RNA isolation. Incubation of RNA at high temperatures has been shown to increase reversal and removal of mono-methylol groups from RNA bases increases performance in downstream applications (Masuda et al., 1999). It may be that inclusion of this step in the Roche method would increase RNA yield. Whereas the Qiagen kit and the Tri Reagent gave good A260/230 ratios indicating minimal contamination the Roche High Pure FFPE Micro RNA kit gave very low A260/230 ratios. This could be as a result of incomplete removal of the glassﬁbres which can interfere with absorbance at 260 nm.

The isolation method did not affect the RIN value of RNA isolated.

The degree of degradation we observed was comparative with those seen in other studies (Reis et al., 2011). In fact Madabusi et al.

(2006)have shown that RNA with a RIN value as low as 1.4 can be successfully used for gene expression analysis. A number of studies have shown that there is a good correlation in gene expression

0 5 10 15 20 25 30

***

*

**

18S

Ct

0 5 10 15 20 25 30

Ct

-Actin

***

*

***

0 5 10 15 20 25 30 35

GAPDH

Ct

***

*

***

β

Fig. 4.Average threshold cycle (Ct) values for housekeeping genesβ-actin, 18S and GAPDH using cDNA generated using three different reverse transcriptase (RT) enzymes in a SYBR Green qRT-PCR reaction. Either MMLV with reduced RNaseH activity (MMLV/H⁻), thermostable MMLV (MMLV/TS) or wild-type MMLV (MMLV) was used (n = 6 for each). Error bars represent standard error of the mean. * = p-valueb0.05, ** = p-valueb0.01, *** = p-valueb0.001.

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between fresh frozen tissue compared with FFPE tissue despite ex- tensive RNA degradation and that they can be successfully used in downstream applications such as qRT-PCR (Dunn et al., 2009).

A number of varieties of M-MLV RTs are now commercially available for cDNA synthesis. The M-MLV/H⁻lacks RNaseH activity and is anin-vitroevolution of the wild-type M-MLV which has endogenous RNaseH activity. RNaseH competes with the polymerase activity against the RNA template and the DNA primer resulting in decreased formation of the RNA template-primer complex and lower yields cDNA. The removal of RNaseH activity has been shown to improve thermostability, processivity and an increased synthesis rate compared to wild-type M-MLV (Gerard et al., 1989). An M-MLV with increased thermostability (M-MLV/TS) is available from Bioline and exhibits high stability at higher temperatures. Increased thermostability allows the RT reaction to proceed at a higher temperature which can prevent RT inhibition of secondary structures present in the RNA. We assessed the effect of variants of M-MLV enzymes on cDNA synthesis yield and the size of ampliﬁable DNA products using RNA isolated from both the Qiagen kit and Tri Reagent using RT-PCR and found the average ampliﬁable length of the RNA to be ~ 200 bp.

This is in accordance with other studies that have shown that the average size of fragmented RNA isolated from FFPE samples is about 200 bp as estimated from formaldehyde-agarose gel electrophoresis (Antonov et al., 2005; Chen et al., 2007; Lehmann and Kreipe, 2001;

Lewis et al., 2001). Tri Reagent FFPE RNA performed poorer when try- ing to amplify 212 bp PCR products compared with the Qiagen

extracted RNA independent of the RT enzyme used. This may be a result of increased PCR inhibition carried over by RNA isolated using Tri Reagent such as guanidine thiocyanate, phenol, ethanol or proteinase K, all of which can affect the RT conversion step (Bustin and Nolan, 2004). Alternatively it may be that RNA isolated from the Qiagen kit had a greater degree of cross linking reversal and decreased RNA fragmentation. An additional RNA clean up step and puriﬁcation of the RT reaction prior to qRT-PCR may augment detection of longer PCR products when using Tri Reagent FFPE RNA.

Next we assessed the effect of RT enzyme on mRNA expression detection using qRT-PCR. The M-MLV/H⁻RT enzyme resulted in a significantly lower Ct value for all genes indicative of the greater limit of detection and sensitivity of gene detection. The M-MLV/H⁻RT enzyme has a higher resistance to qRT-PCR inhibitors which may be present when using FFPE derived cDNA (Arezi et al., 2010). In addition we found that as amplicon length increased the reliability and linear regression of a standard curve dilution decreased. We do not believe that this is a direct effect of qRT-PCR efficiency as when the slope of the standard curve was used to calculate PCR primer efficien- cies there was no difference between the genes used (β-actin: 93%, 18S: 98% efficiency and GAPDH: 96%). This suggests that one of the most important factors for consideration when using RNA from archival sources is the amplicon size of the gene to be analysed by qRT-PCR. We further assessed the effect of RT on mRNA expression detection of target gene expression between a benign prostate sample and a prostate carcinoma sample set using CDH1 and CDH2 0

200 400 600 800 1000 1200

Benign Cancer

MMLV, H- MMLV,TS MMLV

CDH1/-Actin

CDH1/E-Cadherin

0 500 1000 1500 2000 2500

Benign Cancer

CDH2/N-Cadherin

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Benign Cancer

MMLV, H- MMLV,TS CDH1/CDH2 (Normalised to -Actin) MMLV

A B

C

β CDH2/-Actinβ

β

Fig. 5.Fold change in mRNA expression for A) E-Cadherin (CDH1), B) N-Cadherin (CDH2) and C) ratio ofCDH1toCDH2between benign and cancer sample (n = 6 for each). Each benign and cancer sample had cDNA generated using either MMLV with reduced RNaseH activity (MMLV/H⁻), thermostable MMLV (MMLV/TS) or wild-type MMLV (MMLV) (n= 6).

Error bars represent standard error of the mean. * = p-valueb0.05, ** = p-valueb0.01, *** = p-valueb0.001.

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targeted primers and we found that the RT enzyme can have dra- matic effects on fold changes and ratio of gene expression observed.

This is important as the only difference is the RT enzyme, all other parameters are the same, and this result suggests that the choice of RT enzyme within a lab or a cohort sample set can drastically affect the reported outcomes. This could also affect the ability to reliably reproduce gene expression results between labs or different co- horts and thus the RT enzyme should be considered when interpreting and/or validating results.

In conclusion, our RT-PCR data shows that biologically assayable RNA can be isolated from FFPE prostate tissue and it is suitable for qRT-PCR analysis if thefinal product of amplification is designed to be approximately 200 bp or less. Based on the ease of use and consistent results obtained we would also recommend the use of the Qiagen FFPE RNeasy kit when isolating RNA from FFPE materials. Other studies have also demonstrated the consistent and reliable use of the Qiagen FFPE RNeasy kit in isolating high yield biologically relevant RNA (Boeckx et al., 2011; Deben et al., 2013). We also show that a M-MLV RT enzyme with reduced RNaseH activity gave significantly increased cDNA yield and increased qRT-PCR sensitivity when using FFPE RNA from prostate tissue. The relative performance of the RT enzymes from other tissue types may vary and should be evaluated prior to any study. Given the influence of the RT enzyme on the RNA to cDNA conversion capacity of FFPE material and the evaluation of RT sensitivity and limits we recommend that it is useful to perform a pilot study to optimise the extraction protocol and RT to maximise success in downstream applications such as qRT-PCR.

Conﬂict of interest

The authors declare that there are no conﬂicts of interests.

Acknowledgments

The authors would like to thank Saima Karamat (North Bristol NHS Bristol Trust) for her technical assistance in cutting the wax sections and Jane Coghill (University of Bristol Genomics Facility) for her technical expertise in the analysis of the RIN values.

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