REVERSE TRANSCRIPTION AND POLYMERASE CHAIN-REACTION TECHNIQUE FOR QUANTIFICATION OF MESSENGER-RNA IN PRIMARY ASTROCYTE CULTURES

The reverse transcription and polymerase chain reaction technique (RT- PCR) was assessed for the quantification of changes in mRNA levels fro m primary astrocyte cultures. The effects of dibutyryl cyclic AMP (dBc AMP) on glial fibrillary acidic protein (GFAP) mRNA and the effects of tumor necrosis factor-alpha (TNF-alpha), interleukin-I beta (IL-1beta ), and lipopolysaccharide (LPS) on interleukin-6 (IL-6) mRNA were exam ined. Two quantitative PCR methods were used: one involved carrying ou t the reaction in the exponential phase and the other involved the coa mplification of a competitive target sequence. Increased GFAP mRNA in response to chronic dBcAMP treatment and increased IL-6 mRNA in respon se to TNF-alpha/IL-1beta were readily detected. Both RT-PCR techniques were found to be suitable for the detection of large as well as small er (twofold) changes in mRNA levels. The advantages and limitations of RT-PCR for mRNA quantification are discussed. (C) 1993 Wiley-Liss, In c.