A general method for quantitative PCR analysis of mRNA levels for members of gene families: application to GABAA receptor subunits.
We have developed a sensitive, PCR-based method for quantitating changes in mRNA levels of members of gene families. In this approach, total mRNA is converted to cDNA and then PCR is carried out on family members simultaneously, using primers derived from regions conserved among family members. This is followed by gel electrophoresis and blotting of the product to filters. The level of expression of individual family members is determined by separate hybridizations using probes unique for each member and derived from sequences between the PCR primers. In this manner the same aliquot of mRNA, the same reverse transcriptase reaction, PCR, gel electrophoresis, and denaturation and blotting are used for analysis of each family member. Thus, experimental variation is minimized, and changes in mRNA levels of family members relative to one another can be monitored with precision. In addition, if a family member is known not to change as a result of the treatment employed, this mRNA can be used to normalize the data from other members and thereby allow individual variations to be quantitated. We have applied this approach to members of the GABAA receptor subunit gene family and studied effects of chronic ethanol treatment on mRNAs corresponding to several GABAA receptor subunits.
Buck
K J
KJ
Dept. of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Harris
R A
RA
Sikela
J M
JM
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D000431:Ethanol / Q000494:pharmacology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D011963:Receptors, GABA-A / Q000032:analysis* / Q000187:drug effects / Q000235:genetics; D015203:Reproducibility of Results
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D000431:Ethanol; D012194:RNA-Directed DNA Polymerase