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 mR...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2003-01-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/03341dd05 |
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| Summary: | 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. |
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| ISSN: | 0736-6205 1940-9818 |