Abstract
A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 524-531 |
| Number of pages | 8 |
| Journal | BioTechniques |
| Volume | 28 |
| Issue number | 3 |
| State | Published - 2000 |
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ASJC Scopus subject areas
- Clinical Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
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Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA. / Swift, Galvin H.; Peyton, Michael J.; MacDonald, Raymond J.
In: BioTechniques, Vol. 28, No. 3, 2000, p. 524-531.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA
AU - Swift, Galvin H.
AU - Peyton, Michael J.
AU - MacDonald, Raymond J.
PY - 2000
Y1 - 2000
N2 - A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.
AB - A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.
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UR - http://www.scopus.com/inward/citedby.url?scp=0034012360&partnerID=8YFLogxK
M3 - Article
C2 - 10723567
AN - SCOPUS:0034012360
VL - 28
SP - 524
EP - 531
JO - BioTechniques
JF - BioTechniques
SN - 0736-6205
IS - 3
ER -