Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide Positions. (January 2003)

• Record Type: Journal Article
• Authors: Stenman, J.; Lintula, S.; Rissanen, O.; Finne, P.; Hedström, J.; Palotie, A.; Orpana, A.
• Is Part Of: Biotechniques. Volume 34:Number 1(2003)
• Page Start: 172
• Page End: 177
• Publication Date: 2003-01
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/03341dd05
• Languages: English
• ISSNs: 0736-6205

Abstract

Accurate analysis of mRNA expression levels of SNPs, highly homologous genes, and splicing variants requires techniques capable of quantifying low-copy-number mRNAs differing at single nucleotide positions. We have used an RT-PCR-based technique based on co-amplification of closely related target mRNA transcripts and assessed the effect of the stochastic distribution of low-copy-number templates on sampling variation when quantifying rare mRNA transcripts. The technique was optimized for maximal sensitivity to enable the analysis of samples containing a subpopulation of target cells and small microdissected samples. We demonstrate that the input level of template molecules is a critical determinant of the achievable assay precision. A minimum of approximately 50 molecules of template is required to discriminate between 2-fold differences in the expression levels of two transcripts. At levels above 1000 molecules of input template, the stochastic effects on sampling variation become negligible.