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Study results from E. Nardon et al provide new insights into molecular pathology
2010 APR 27 - (NewsRx.com) -- "Real time quantitative reverse transcription-PCR (qRT-PCR) is the most sensitive technique for detection and quantification of mRNA targets. Reliable quantification of gene expression in formalin-fixed, paraffin-embedded tissues (FFPE), however, has been subjected to serious limitations so far, mainly due to the fragmentation of RNA transcripts," researchers in Trieste, Italy report. "We tried to improve the sensitivity and reliability of mRNA quantification in FFPE by boosting the reverse transcription (RT) step, that is neglected in most of the protocol analysis, but that represents the first confounding event in a quantitative analysis. For this purpose, we compared yield, reproducibility and linearity of RTs performed with random hexamers, random pentadecamers, or a mixture of antisense specific primers in presence of either Moloney murine leukemia virus (MmLV) or the avian myeloblastosis virus (AMV) enzymes. Random primers were tested at two concentrations, 0.14 and 3.35 nmol/reaction. Our qRT-PCR results indicate an improvement of RT yield when using the highest concentration of random oligos with MmLV (from -1.4 to -4.1 C(t)s) in comparison to the lowest concentration. Moreover, more reliable standard curves and therefore, efficiencies were obtained. RT reactions performed with specific primers and AMV were those with the highest yield, but efficiencies were unreliable, due to the RT enzyme-driven PCR inhibition," wrote E. Nardon and colleagues. The researchers concluded: "Random priming at the 3.35 nmol/reaction concentration seems to be the most convenient strategy in assays using RNA obtained from FFPE tissues." Nardon and colleagues published their study in Experimental and Molecular Pathology (Higher random oligo concentration improves reverse transcription yield of cDNA from bioptic tissues and quantitative RT-PCR reliability. Experimental and Molecular Pathology, 2009;87(2):146-151). For additional information, contact G. Stanta, Cattinara Hospital, Dept. of Clin Morphol & Technol Science, Surg Pathol Bldg, Str Fiume 447, I-34149 Trieste, Italy. Publisher contact information for the journal Experimental and Molecular Pathology is: Academic Press Inc. Elsevier Science, 525 B St., Ste. 1900, San Diego, CA 92101-4495, USA. Keywords: City:Trieste, Country:Italy, Antisense Technology, Biotechnology, Gene Therapy, Life Sciences, Molecular Medicine, Pathology, Treatment This article was prepared by Life Science Weekly editors from staff and other reports. Copyright 2010, Life Science Weekly via NewsRx.com.
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