title: A M-MLV reverse transcriptase with reduced RNaseH activity allows greater sensitivity of gene expression detection in formalin fixed and paraffin embedded prostate cancer samples.
abstract: Formalin fixed and paraffin embedded (FFPE) human tissue collections are an invaluable resource for retrospective gene expression studies. However formalin fixation 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 (<200bp) 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.
authors.affiliation: Faculty of Health and Life Sciences, University of the West of England, Bristol, UK. Rachel.Hagen@uwe.ac.uk
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Hagen
authors.forename: Anthony
authors.initials: A
authors.lastname: Rhodes
authors.forename: Jon
authors.initials: J
authors.lastname: Oxley
authors.forename: Michael R
authors.initials: MR
authors.lastname: Ladomery
mesh_terms: D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D016612:Paraffin Embedding; D011471:Prostatic Neoplasms / Q000235:genetics*; D012334:RNA, Neoplasm / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016914:Ribonuclease H / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D012334:RNA, Neoplasm; D005557:Formaldehyde; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H
keywords: CDH1; CDH2; E-Cadherin; FFPE; Formalin fixed paraffin embedded; M-MLV; MMLV; Moloney Murine Leukemia Virus; N-Cadherin; PCR; RT; RT-PCR; Reverse transcriptase; qRT-PCR; quantitative real-time PCR; reverse transcriptase; reverse transcriptase PCR