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

• Published in: Experimental and molecular pathology 2013-08, Vol.95 (1), p.98-104
• Main Authors: Hagen, Rachel M., Rhodes, Anthony, Oxley, Jon, Ladomery, Michael R.
• Format: Article
• Language: English
• Subjects: FFPE; Formaldehyde; Gene Expression Profiling - methods; Humans; Limit of Detection; M-MLV; Male; Moloney murine leukemia virus - enzymology; Paraffin Embedding; PCR; Prostatic Neoplasms - genetics; Real-Time Polymerase Chain Reaction - methods; Reverse transcriptase; Reverse Transcriptase Polymerase Chain Reaction - methods; Ribonuclease H - metabolism; RNA, Neoplasm - isolation & purification; RNA-Directed DNA Polymerase - metabolism; Sensitivity and Specificity
• ISSN: 0014-4800; 1096-0945
• DOI: 10.1016/j.yexmp.2013.05.008

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 (