Computational protocol: Adhesion GPCRs are widely expressed throughout the subsections of the gastrointestinal tract

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Protocol publication

[…] The RT-qPCR was performed using a MyiQ thermal cycler (Bio-Rad Laboratories, Sweden). Each RT-qPCR reaction, with a total volume of 20 μl, contained cDNA synthesized from 25 ng of total RNA, 0.25pmol/μl of each primer, 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 4 mM MgCl2, 0.2 mM NTP, SYBR Green (1:50000) (Invitrogen, USA) and 0.02U/μl Taq DNA polymerase (Biotools, Spain). The reaction conditions were the following: initial denaturation at 95°C for 4 min, succeeded by 40 cycles at 95°C for 15 s, 55–62°C for 30 s (optimal annealing temperature) and 72°C for 30 s. This was followed by 81 cycles at 55°C for 10 s (increased by 0.5°C per cycle). All real-time PCR experiments were run in triplicates. A negative control for each primer pair and a positive control with 25 ng of rat genomic DNA was included on each plate. [...] Bio-Rad iQ5 software v2.0 software (Bio-Rad Laboratories, Sweden) was used to process RT-qPCR data and obtain threshold cycle (Ct) values. Melting curves were analyzed to assure that only one product with the expected melting point was amplified and that this was separate from the negative control. LinRegPCR was used to calculate PCR efficiencies for each sample and Grubbs' test (GraphPad, USA) was applied to exclude any outliers when calculating the average PCR efficiency for each primer pair. The delta Ct method [] was used to convert Ct values into relative quantities with the standard deviation, and the highest expression was normalized to 1. The GeNorm software [] was used with results from the five most stable housekeeping genes to calculate normalization factors for each tissue to compensate for differences in cDNA quantity. Subsequently, the normalized quantities were calculated and maximum expression was set to 1: all relative expression values are shown as fold decrease with respect to the detected maximum expression (see Additional file ). […]

Pipeline specifications

Software tools Biotools, LinRegPCR
Applications Population genetic analysis, qPCR
Organisms Rattus norvegicus