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.
Kelleher
K L
KL
Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia. kim.kelleher@anu.edu.au
Leck
K J
KJ
Hendry
I A
IA
Matthaei
K I
KI
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