Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA.
Biotechniques
; 28(3): 524, 526, 528, 530-1, 2000 Mar.
Article
en En
| MEDLINE
| ID: mdl-10723567
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN Mensajero
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
Límite:
Animals
Idioma:
En
Revista:
Biotechniques
Año:
2000
Tipo del documento:
Article
País de afiliación:
Estados Unidos