Quantitative detection of low-copy-number mRNAs differing at single nucleotide positions.
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.
Stenman
J
J
Dept. of Clinical Chemistry, Helsinki University Central Hospital, FIN-00029 HUS, Helsinki, Finalnd. jakob.stenman@hus.fi
Lintula
S
S
Rissanen
O
O
Finne
P
P
Hedström
J
J
Palotie
A
A
Orpana
A
A
D000951:Antigens, Neoplasm / Q000235:genetics; D018628:Gene Dosage; D006801:Humans; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018401:Sample Size; D012680:Sensitivity and Specificity; D015843:Serpins; D013269:Stochastic Processes
D000951:Antigens, Neoplasm; D012333:RNA, Messenger; D015843:Serpins; C051072:squamous cell carcinoma-related antigen