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Reverse transcription and polymerase chain reaction technique for quantification of mRNA in Primary astrocyte cultures

✍ Scribed by G. M. Murphy Jr.; X. -C. Jia; A. C. H. Yu; Y. L. Lee; J. R. Tinklenberg; Dr. L. F. Eng

Publisher: John Wiley and Sons
Year: 1993
Tongue: English
Weight: 1005 KB
Volume: 35
Category: Article
ISSN: 0360-4012
DOI: 10.1002/jnr.490350607

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✦ Synopsis

The reverse transcription and polymerase chain reaction technique (RT-PCR) was assessed for the quantification of changes in mRNA levels from primary astrocyte cultures. The effects of dibutyryl cyclic AMP (dBcAMP) on glial fibrillary acidic protein (GFAP) mRNA and the effects of tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-lp), and lipopolysaccharide (LPS) on interleukin-6 (IL-6) mRNA were examined. Two quantitative PCR methods were used: one involved carrying out the reaction in the exponential phase and the other involved the coamplification of a competitive target sequence. Increased GFAP mRNA in response to chronic dBcAMP treatment and increased IL-6 mRNA in response to T N F d I L -l P were readily detected. Both RT-PCR techniques were found to be suitable for the detection of large as well as smaller (twofold) changes in mRNA levels. The advantages and limitations of RT-PCR for mRNA quantification are discussed.

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