Reverse transcription and polymerase chain reaction technique for quantification of mRNA in primary astrocyte cultures.
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-alpha), interleukin-1 beta (IL-1 beta), 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-alpha/IL-1 beta 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.
Murphy
G M
GM
Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, California.
Jia
X C
XC
Yu
A C
AC
Lee
Y L
YL
Tinklenberg
J R
JR
Eng
L F
LF
D000818:Animals; D001253:Astrocytes / Q000737:chemistry* / Q000378:metabolism; D001345:Autoradiography; D015415:Biomarkers; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D007375:Interleukin-1 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D015415:Biomarkers; D005904:Glial Fibrillary Acidic Protein; D007375:Interleukin-1; D008070:Lipopolysaccharides; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003994:Bucladesine; D012194:RNA-Directed DNA Polymerase