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A synthetic standard for competitive RT-PCR quantitation of 13 GABA receptor type A subunit mRNAs in rats and mice
- Source :
- Journal of Neuroscience Methods. 85:89-98
- Publication Year :
- 1998
- Publisher :
- Elsevier BV, 1998.
-
Abstract
- We describe a synthetic 769-bp DNA internal standard, GABARQuant 1, for measuring mRNAs of 13 GABA A receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR). When it is transcribed into cRNA, added in known amounts to target mRNAs in extracts from rat or mouse tissue, competitively reverse transcribed into cDNA, and amplified by the polymerase chain reaction (PCR), the relative intensities of the amplified, stained target and standard DNA bands enable measurement of small amounts of mRNAs for GABA A receptor subunits α 1–6, β 1–3, γ 1–3 and δ and the three cellular markers β -actin, light neurofilament protein, and glutamine synthetase. For the subunits, most standard products (263–504 bp) differ in size from target products (398–564 bp) by 10–20%. Primer pairs span at least one intron, to prevent interference by genomic DNA, and at least one rat versus mouse restriction fragment length polymorphism (RFLP), to enable rat products to be distinguished from mouse products.
- Subjects :
- Reverse Transcriptase Polymerase Chain Reaction
General Neuroscience
Protein subunit
Biology
Receptors, GABA-A
Molecular biology
Reverse transcriptase
Rats
Mice
genomic DNA
chemistry.chemical_compound
Real-time polymerase chain reaction
Biochemistry
chemistry
Complementary DNA
Animals
RNA, Messenger
Primer (molecular biology)
Restriction fragment length polymorphism
Polymorphism, Restriction Fragment Length
DNA
Subjects
Details
- ISSN :
- 01650270
- Volume :
- 85
- Database :
- OpenAIRE
- Journal :
- Journal of Neuroscience Methods
- Accession number :
- edsair.doi.dedup.....ff613c7104a9cc7d9217e9930a8a94b0