By Brinda K. Rana, Paul A. Insel (auth.), Anthony P. Davenport (eds.)
Maintaining the excessive criteria set by means of the winning first version, Anthony P. Davenport and a panel of hands-on researchers from the pharmaceutical and academia benefit from the most recent advancements to supply targeted useful equipment for learning receptors in silico, in vitro, and in vivo. those with no trouble reproducible innovations disguise mining from curated databases, settling on novel receptors through excessive throughput screening, molecular easy methods to establish mRNA encoding receptors, radioligand binding assays and their research, quantitative autoradiography, and imaging receptors via positron emission tomography (PET). Highlights comprise phenotypic characterization of receptors in knock-out mice, imaging receptors utilizing eco-friendly fluorescent protein and fluorescent resonance strength move, and quantitative research of receptor mRNA by means of TaqMan polymerase chain response (PCR). those ligand binding innovations are ideal for exploring the remarkable variety of new receptor platforms now rising and the so-called "orphan" receptors whose activating ligand has now not been pointed out. The protocols keep on with the winning equipment in Molecular Biology™ sequence layout, every one providing step by step laboratory directions, an advent outlining the main at the back of the approach, lists of the required apparatus and reagents, and tips about troubleshooting and warding off identified pitfalls.
entire and cutting-edge, Receptor Binding ideas, moment variation deals educational and advertisement researchers within the pharmaceutical and biotechnology industries a suite of confirmed concepts for the profitable characterization of receptors and the phenotyping of transgenic animals, together with knock-outs.
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Additional info for Receptor Binding Techniques
It is the recommendation of the authors that all investigators should do this. Alternatively, if DNA contamination of an RNA sample is suspected, this can be verified by gel electrophoresis. However, given the sensitivity of PCR approach, DNase-verification by TaqMan PCR is the preferred method (especially when dealing with GPCRs, which are typically expressed with low abundance). When an RNA sample is run out on a denaturing agarose gel, two discrete bands (18S and 28S RNA) should be observed.
During the exponential phase of PCR, the amount of product is proportional to the initial copy number of the template. This relationship changes as the rate of amplification approaches a plateau (hence thresholds should be set within the exponential phase of PCR). Once the threshold is defined, the CT (threshold cycle) values and copy numbers (if standards are present on the plate) can be exported for statistical data analysis. As noted above, to assess RNA quality, template loading efficiency and cDNA conver- Quantitating GPCR Expression by TaqMan® PCR 45 Fig.
When isolating RNA, work quickly and ensure that all tubes, solutions/reagents and pipet tips used are RNase free. b. RNA is subject to degradation under conditions of excessive shear stress, so exercise caution when drawing RNA-containing fluids into pipets (because large shearing forces can be generated in pipet tips with small openings). 3. In order to provide standardization across samples, all RNA samples of interest should be reverse-transcribed into cDNA templates at the same time. Note also that, although the RNA quantity utilized in the protocol is an optimal amount for the reverse transcription reaction, the method will efficiently reverse transcribe down to 50 ng/µL and up to 1µg/µL RNA (or 10 µg/µL total RNA).
Receptor Binding Techniques by Brinda K. Rana, Paul A. Insel (auth.), Anthony P. Davenport (eds.)