Quantification of G protein mRNA using reverse transcription and competitive PCR with a colorimetric microplate assay.
This paper reports an assay for the quantification of levels of specific mRNA for the alpha subunits of the inhibitory G proteins G alpha i-1, G alpha i-2, and G alpha i-3. The assay employs reverse transcription and competitive polymerase chain reaction (PCR) coupled to enzyme-linked oligonucleotide sorbent assay for differential detection and quantification of PCR products. The assay was conducted with conventional thermal block PCR cyclers as well as rapid air microcapillary cyclers. The detection stage consists of three steps using synthetic oligonucleotides, commercially available reagents and a conventional 96-well plate absorbance reader at settings of 450 and 630 nm. The assay is: (1) rapid, requiring about 3 h for quantification of PCR products; (2) safe, being non-radiometric; (3) relatively simple; (4) highly sensitive, being capable of detecting less than 10 initial copies of target cDNA; (5) precise, resolving two-fold differences in initial copy numbers of specific sequences as low as 10(-20) mol; (6) linear over a 3 log range, with two-fold differences in the quantity of cDNA producing consistent reductions in quantity of specific cDNA detected; and (7) reproducible, intra-assay and inter-assay coefficients of variation being 11.9 and 14.7%, respectively.
Mokkapatti
R
R
Department of Pharmacology, University of Pittsburgh, Pennsylvania 15261, USA.
Conn
R A
RA
Carcillo
J A
JA
Romkes
M
M
Jackson
E K
EK
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D019206:GTP-Binding Protein alpha Subunits, Gi-Go