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Use of internally controlled reverse transcriptase‐polymerase chain reaction for absolute quantitation of individual multidrug resistant gene transcripts in tissue samples
Author(s) -
Zhang Fang,
Riley Joan,
Gant Timothy W.
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170144
Subject(s) - reverse transcriptase , multiple drug resistance , biology , polymerase chain reaction , rnase p , messenger rna , rnase h , real time polymerase chain reaction , microbiology and biotechnology , gene , reverse transcription polymerase chain reaction , nuclease protection assay , polymerase , rna , drug resistance , genetics , rna dependent rna polymerase
Research into the relative importance of P‐glycoprotein (pgp) overexpression, in comparison to other mechanisms of multidrug resistance (mdr) phenotype development, has been hampered by difficulties of measurement in clinical samples. The small size, heterogeneity and often poor quality of clinical specimens renders the quantitation of mdr mRNA species by Northern analysis or RNAse protection difficult. The reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay has both the sensitivity and specificity to make it suitable for the analysis of mdr mRNA levels in clinical samples. It also has a significant speed advantage over Northern and RNAse protection analysis. Unfortunately the variable nature of the reactions involved have made it difficult to obtain accurate and reproducible quantitative data. To overcome this difficulty we constructed internal RNA standards to each human and rat mdr mRNA species. When included in the RT‐PCR reaction these internal standards allow both comparative and absolute quantitation of individual mdr family mRNA levels.