Low efficiency of the Moloney murine leukemia virus reverse transcriptase during reverse transcription of rare t(8;21) fusion gene transcripts.
The resolving power of RT-PCR is limited by the efficiency of RNA-to-cDNA conversion. Methods to determine this efficiency, using a real-time PCR assay for quantifying AML1-MTG 8 [t(8;21)] fusion gene transcripts, are described. The efficiency is shown to be directly proportional to RNA template levels. The Moloney murine leukemia virus (MMLV) reverse transcriptase enzyme's conversion efficiency was calculated to be approximately 20%. The efficiency was even lower (6%) when target templates were rare (single molecules) in the RT reactions. Levels of nonspecific or background RNA present in the RT reaction reduced the reverse transcriptase's conversion efficiency. This background effect was particularly pronounced when the specific template was present in rare amounts.
Curry
John
J
Department of Immunology, Molecular and Cellular Biology, University of California, Berkeley 94720-3200, USA. curry@uclink4.berkeley.edu
McHale
Cliona
C
Smith
Martyn T
MT
D050676:Core Binding Factor Alpha 2 Subunit; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
C107844:AML1-ETO fusion protein, human; D050676:Core Binding Factor Alpha 2 Subunit; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors; D012194:RNA-Directed DNA Polymerase