0000000000158256
AUTHOR
Martina Ficková
Quantitation of GABA transporter 3 (GAT3) mRNA in rat brain by competitive RT-PCR.
Gamma-amino butyric acid is the major inhibitory neurotransmitter in the brain. GABA transporters (GATs) remove GABA from the synaptic cleft. Till now, five distinct GABA transporters have been cloned and termed consecutively GAT1 to GAT4 and vGAT. To study the mechanisms by which tolerance and dependence associated with drugs enhancing GABAergic transmission is brought upon we analysed the mRNA expression levels of GATs in various brain regions under different conditions. In this paper, we describe our protocol for measurement of GAT3 mRNA expression, and its validation through control experiments for the various steps. We performed competitive reverse transcription and polymerase chain re…
Molecular cloning of rat G-protein-coupled receptor kinase 6 (GRK6) from brain tissue, and its mRNA expression in different brain regions and peripheral tissues
The rat G-protein-coupled receptor kinase 6 (GRK6) cDNA was cloned from rat brain tissue by a combination of reverse-transcription polymerase chain reactions (RT-PCR), based on homology to the cloned human GRK6, and rapid amplification of cDNA ends (RACE-PCR). We obtained a clone of 2817 bp with an open reading frame of 1731 bp encoding for a protein of 576 amino acids that is 96.7% identical and 97.9% similar to its human counterpart. mRNA was detectable in all brain areas examined. In addition, GRK6 was expressed in skeletal muscle, small intestine, aorta, liver, heart, lung, thymus, stomach, uterus and kidney.
Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase 6: an improved method to determine 5′- and 3′-cDNA ends
Abstract Rapid cloning of 5′- and 3′-cDNA ends polymerase chain reaction (5′-/3′-RACE-PCR) is useful to determine unknown 5′- and 3′-cDNA termini. Even if the method can yield complete cDNA sequences within a couple of days, the RACE procedure bears some characteristic traps and often results in amplification of unspecific PCR-products. Here we used improved 5′- and 3′-RACE-PCR protocols to obtain the complete cDNA sequence of the G-protein-coupled receptor kinase 6 (GRK6) from a rat brain cDNA library. The use of an anchored oligo-(dT) 16 -V-primer in the cDNA synthesis, the addition of single-sided PCR steps prior to the RACE-PCRs and the optimization of the dA-tailing reaction conditions…