Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction
Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for...
| Veröffentlicht in: | BioTechniques. - 1993. - 56(2014), 6 vom: 13. Juni, Seite 319-25 |
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| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't RNA alternative splicing ddPCR droplet digital PCR hTERT qPCR real-time PCR telomerase mehr... |
| Zusammenfassung: | Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for the simultaneous quantification of multiple alternatively spliced RNA transcripts, such as in the case of hTERT. Here we show droplet digital PCR (ddPCR) provides sensitive, simultaneous digital quantification in a single reaction of two alternatively spliced single deletion hTERT transcripts (α-/β+ and α+/β-) as well as the opportunity to manually quantify non-deletion (α+/β+) and double deletion (α-/β-) transcripts. Our ddPCR method enables direct comparison among four alternatively spliced mRNAs without the need for internal standards or multiple primer pairs specific for each variant as real-time PCR (qPCR) requires, thus eliminating potential variation due to differences in PCR amplification efficiency |
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| Beschreibung: | Date Completed 26.01.2015 Date Revised 21.10.2021 published: Electronic-eCollection Citation Status MEDLINE |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/000114179 |