A one-step quantitative reverse transcription polymerase chain reaction procedure.
Our laboratory has developed a one-step quantitative reverse transcription polymerase chain reaction (RT-PCR) procedure in which the reverse transcriptase enzyme and Taq DNA polymerase are combined in the one tube and a single, non-interrupted, thermal cycling program is performed. In the past, RT-PCR has been carried out with two separate steps: (1) reverse transcription of RNA to generate a cDNA pool and (2) polymerase chain reaction amplification of the cDNA. The two-step method can affect the accuracy of the procedure as the total number of manipulations is greater, thereby allowing a greater chance for pipetting errors. Quantitation by our method is achieved in a single reaction by the use of a competitive internal standard that is identical in sequence to the target RNA except for a deletion of 107 base pairs and uses identical primers and cycling conditions. Using this method, we have been able to quantify the amount of message of a G protein (G(zalpha)), in small amounts of tissue, such as dorsal root ganglia, from embryonic as well as postnatal mice.
Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia. kim.kelleher@anu.edu.au
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Kelleher
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Leck
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Hendry
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Matthaei
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D019204:GTP-Binding Proteins / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
D017931:DNA Primers; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D019204:GTP-Binding Proteins