The use of reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate specific gene expression in multidrug-resistant cells.
Expression of specific genes at the level of mRNA can be studied using techniques such as Northern blot, slot/dot blot, RNase protection assay, in situ hybridisation and RT-PCR. In this article these methods of analysis are compared; RT-PCR offers higher levels of specificity and sensitivity than traditional methods of RNA analysis and as such has become the method of choice for the study of gene expression. The RT-PCR technique is described in detail with sections dealing with RNA extraction, choice of primers (including the use of cDNA sequence data bases), PCR and RT-PCR protocols in addition to the limitations of the method. The study of one particular mRNA transcript (MDR1) using RT-PCR is discussed in detail. Recently described methods for quantitation of PCR products are discussed. Quantitative PCR would appear to offer a method of studying gene expression in a more extensive way than has been possible to date.
O'Driscoll
L
L
National Cell and Tissue Culture Centre/Bioresearch Ireland, Dublin City University.
Daly
C
C
Saleh
M
M
Clynes
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins / Q000096:biosynthesis; D000818:Animals; D016477:Artifacts; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D004351:Drug Resistance / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D001613:beta 2-Microglobulin / Q000096:biosynthesis
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D002352:Carrier Proteins; D017931:DNA Primers; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; D012194:RNA-Directed DNA Polymerase; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; C020118:ESD protein, human