Computational protocol: Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)

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Protocol publication

[…] Sequences of the 13 candidate cetacean HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were obtained from bottlenose dolphin, striped dolphin, beluga whale, killer whale and fin whale (Balaenoptera physalus) from GenBank ( and ). Besides 11 HKGs have been evaluated or used in previous studies (; ; ; ; ; ; ; ; ; ), the other 2 genes that could participate in other different cell functions were also included (; ). Primers and corresponding UPL probes were designed and chosen using Roche UPL design software (ProbeFinder, v.2.49) based on Primer3 software (). All designed primer pairs were checked by in silico PCR algorithm in ProbeFinder, which searches the relevant genome and transcriptome for possible mis-priming sites for either of the PCR primers. Before qPCR experiment, the specificity of primers of 13 candidate genes was confirmed using Fast-Run Hotstart PCR kit (Protech) and electrophoresis. [...] Corrected Cq values (Cq corr) were transformed from raw Cq values using ΔCq formula, Cq corr = Cqmin − log2E−ΔCq, modified from , where ΔCq is the Cq value of a certain sample minus the Cq value of the sample with the highest expression (lowest Cq, Cqmin) of each HKG as calibrator. Stability of all HKGs were evaluated and ranked using algorithms geNorm (), NormFinder (), comparative ΔCt method () and Bestkeeper () based on a web-based analysis tool RefFinder ( (). RefFinder calculated the geometric mean based on rankings obtained from each algorithm and provides the final comprehensive ranking. Thirty samples were randomly selected from the 60 samples, and the results of HKG ranking using 30 and 60 samples were analyzed comparatively. […]

Pipeline specifications

Software tools Primer3, In-Silico PCR, NormFinder, BestKeeper
Application qPCR
Organisms Huso huso