Computational protocol: Molecular analysis of CIB4 gene and protein in Kermani sheep

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Protocol publication

[…] Tissues including brain, heart, lung, spleen, kidney, liver, ovary, and testis (3 repeats from each tissue) were collected from Kermani sheep (4 males and 2 females) after slaughter. Tissue samples were immediately frozen in liquid nitrogen and stored at –80°C.Total RNA was isolated from each tissue sample using a One Step RNA Reagent Kit (Biobasic Co. Ltd., Iran). The RNA concentration was assessed by spectrophotometry at 260 nm, and RNA quality was assessed by the absorbance 260nm:280nm ratio and electrophoresis on 2% agarose gel stained with ethidium bromide.RNAs were reverse transcribed with RerertAid™ H Minus First Strand cDNA Synthesis Kit (#K1631, Fermentase Co., Iran) and an oligo d(T) primer was used according to manufacturer's protocol. An input of 1 μg total RNA was used for the reaction.Primers 5′-CATGGGGCAATGTCTGAGGT-3′ and 5′-GGTATTTGTGTTCACGTCAAC-3′ for CIB4 gene and 5′-CTGCTGACGCTCCCATGTTTGT-3′ and 5′-CTGCTGACGCTCCCATGTTTGT-3′ for GAPDH gene used for RT-PCR were synthesized by Bioneer Co. (Iran). GAPDH was used to normalize the gene expression data as an endogenous control in the quantitative analysis of RT-PCR, since in some experimental systems its expression is very constant.Samples were amplified using power SYBR Green PCR Master Mix (Iran). All reactions were performed with optical 96-well skirted microplates. Reactions were carried out in a volume of 15 µL consisting of 2X SYBR Green PCR Master Mix, 7.5 µL; template cDNA, 1.5 µL; 10 µM forward and reverse primers, 1 µL; ROX, 0.3 µL and ddH2O, 4.7 µL. PCR protocol was done at 94°C for 5 min, then 40 cycles of 94°C for 30 s, 59°C for 60 s, and 72°C for 45 s and final extension at 72°C for 1 min. To exclude the contamination of unspecific PCR products such as primer dimers, melting curve analysis was generated to all final PCR products.PCR efficiency (E) was estimated for each primer pair, standard curve arbitrary units were set and dilutions of 1, 0.1, 0.01, and 0.001 were made. Fold change in gene expression was calculated using Pfaffl method (). PCR products of CIB4 gene from Kermani sheep were purified from gel using QIAquick Gel Extraction Kits (Qiagen, Iran) and sent to Bioneer Co. (South Korea) for sequencing. Comparative analyses of the nucleotide sequences were performed online at NCBI (http://www.ncbi.nlm.nih/gov) and the phylogenetic tree was constructed by MEGA4.1 (). Predictions of open reading frames and theoretical molecular weights of deduced polypeptides were made by the protein property calculator (http://www.basic.northwestern.edu/biotools/proteincalc.html). The protein isoelectric point was predicted (http://isoelectric.ovh.org/). The domain of Kermani sheep CIB4 protein was predicted with SMART software (http://smart.embl.de/). Prediction of protein characteristics and three dimensional structures were provided by Molecular Bioinformatics Center of National Chiao Tung University (http://ps2v2.life.nctu.edu.tw). The STRING program was used for representing predicted protein interactions (http://string-db.org/version_10). […]

Pipeline specifications

Software tools MEGA, Biotools
Applications Phylogenetics, Population genetic analysis
Organisms Homo sapiens, Ovis aries, Bos taurus, Capra hircus, Camelus dromedarius, Equus caballus, Canis lupus familiaris, Mus musculus
Chemicals Calcium, Nucleotides