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 |
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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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| author | J. Stenman S. Lintula O. Rissanen P. Finne J. Hedström A. Palotie A. Orpana |
| author_facet | J. Stenman S. Lintula O. Rissanen P. Finne J. Hedström A. Palotie A. Orpana |
| author_sort | J. Stenman |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-b0b21cff41794516a7c25dedb262f0d4 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2003-01-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-b0b21cff41794516a7c25dedb262f0d42025-08-20T02:25:50ZengTaylor & Francis GroupBioTechniques0736-62051940-98182003-01-0134117217710.2144/03341dd05Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide PositionsJ. Stenman0S. Lintula1O. Rissanen2P. Finne3J. Hedström4A. Palotie5A. Orpana61Helsinki University Central Hospital, Helsinki, Finland1Helsinki University Central Hospital, Helsinki, Finland1Helsinki University Central Hospital, Helsinki, Finland1Helsinki University Central Hospital, Helsinki, Finland1Helsinki University Central Hospital, Helsinki, Finland1Helsinki University Central Hospital, Helsinki, Finland1Helsinki University Central Hospital, Helsinki, FinlandAccurate 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.https://www.future-science.com/doi/10.2144/03341dd05 |
| spellingShingle | J. Stenman S. Lintula O. Rissanen P. Finne J. Hedström A. Palotie A. Orpana Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide Positions BioTechniques |
| title | Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide Positions |
| title_full | Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide Positions |
| title_fullStr | Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide Positions |
| title_full_unstemmed | Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide Positions |
| title_short | Quantitative Detection of Low-Copy-Number mRNAs Differing at Single Nucleotide Positions |
| title_sort | quantitative detection of low copy number mrnas differing at single nucleotide positions |
| url | https://www.future-science.com/doi/10.2144/03341dd05 |
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