A one-step quantitative reverse transcription polymerase chain reaction procedure

Kim L. Kelleher, Kwong Joo Leck, Ian A. Hendry, Klaus I. Matthaei

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9 Citations (Scopus)

Abstract

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), in small amounts of tissue, such as dorsal root ganglia, from embryonic as well as postnatal mice.

Original languageEnglish
Pages (from-to)100-107
Number of pages8
JournalBrain Research Protocols
Volume6
Issue number3
DOIs
Publication statusPublished - 1 Jan 2001
Externally publishedYes

Keywords

  • Competitive internal standard
  • G proteins
  • Mouse
  • mRNA developmental expression
  • Nervous system
  • Quantitative RT-PCR

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