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Reverse transcription and polymerase chain reaction technique for quantification of mRNA in Primary astrocyte cultures
Author(s) -
Murphy G. M.,
Jia X. C.,
Yu A. C. H.,
Lee Y. L.,
Tinklenberg J. R.,
Eng L. F.
Publication year - 1993
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490350607
Subject(s) - glial fibrillary acidic protein , messenger rna , microbiology and biotechnology , reverse transcription polymerase chain reaction , astrocyte , tumor necrosis factor alpha , reverse transcriptase , polymerase chain reaction , biology , real time polymerase chain reaction , chemistry , biochemistry , immunology , gene , endocrinology , immunohistochemistry , central nervous system
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‐α), interlekin‐1 beta (IL‐1β), 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 TNF‐α/IL‐1β 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. © 1993 Wiley‐Liss, Inc.