Quantitative real-time (q-RT)-polymerase chain reaction (PCR) for survivin mRNA expression

Total RNA was isolated using TRIzol^® ready to use solution procured from M/s Invitrogen (USA) and used as per manufacturer’s recommendations. Complementary DNA (catalog no. 3B 120, biotools B and M Labs, Spain) was prepared for RNA sequences encoding survivin gene of dog using gene-specific primers. Quantitative TaqMan RT-PCR (catalog no. 3B 108, biotools B and M Labs, Spain) assay was carried out for survivin (antiapoptotic protein) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (housekeeping gene) mRNA using the Thermal cycler (EPPENDORF realplex 2.2) instrument according to the manufacturer’s instructions. Published sequences available in the gene bank were used for the designing of required primers for the study. Primers were designed using primer blast (http://www.ncbi.nlm.nih.gov/tools/primerblast/), Genscript^® (https://www.genscript.com/sslbin/app/primer), and primer3plus^® (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi/) sequence analyzing software’s and procured from Bioserve Biotechnologies (India) Pvt Ltd.

The published reference sequence of survivin for dog was from NCBI No: NM-001003348 (Table-1).

Primers and probes used for q-RT-PCR.

q-RT-PCR=Quantitative real-time-polymerase chain reaction, GAPDH=Glyceraldehyde-3-phosphate dehydrogenase

RT-PCR amplification reaction was carried out in a 20 µl reaction mixture containing 10 µl each of mastermix (3B quantimix), and samples were used in duplicate. Relative gene quantification was done by comparative Ct method, and the values were expressed as relative to the reference sample used, as calibrator (Tables-​(Tables-22 and ​and33).

Thermal cycling conditions for amplification of dog GAPDH gene.

GAPDH=Glyceraldehyde-3-phosphate dehydrogenase

Thermal cycling conditions for amplification of dog survivin gene.