Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.
We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.
Vela
J
J
Departamento de Bioquimica, Bromatologia, Toxicologia y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla, C/ Profesor García González s/n, 41012 Seville, Spain.
Vitorica
J
J
Ruano
D
D
D000818:Animals; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D018756:GABA Antagonists / Q000737:chemistry*; D006624:Hippocampus / Q000737:chemistry; D007202:Indicators and Reagents; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D011963:Receptors, GABA-A / Q000737:chemistry*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D018756:GABA Antagonists; D007202:Indicators and Reagents; D012333:RNA, Messenger; D011963:Receptors, GABA-A