Kapa sybr fast qpcr kit master mix 2 abi prism

Relative quantification of gene expression against reference genes GAPDH and RPLPO was performed on a StepOne Real time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), using KAPA SYBR FAST qPCR Kit Master Mix (2×) ABI PRISM (KAPA BIOSYSTEMS, Wilmington, MA, USA) according to manufacturer’s protocol. Primers for all genes were purchased from Eurofins Genomics (Germany), and their sequences are listed in Table S4. The annealing temperature that was used for all the primer pairs was 60 °C. The qPCR program that was selected for all the reactions consisted of the following stages: an initial denaturation stage at 95 °C for 20 s, followed by 40 cycles of amplification (denaturation at 95 °C for 3 s and annealing and extension at 60 °C for 20 s), and, finally, a melt curve stage for each PCR product. All reactions were performed in triplicates. Relative expression of different gene transcripts was calculated by the ΔΔCt method. The Ct value of any gene of interest was normalized to the Ct values of two housekeeping genes (GAPDH and RPLPO).

Total RNA was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from 0.5 ng total RNA using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). KAPA SYBR FAST qPCR Kit Master mix (2×) ABI Prism (Kapa Biosystems, Wilmington, MA, USA) was used as the reaction solution for RT-qPCR. The samples were amplified using a LightCycler 96 System (Roche Diagnostics, Basel, Switzerland). PCR primer sequences are shown in Additional file 1: Table S1. All values were normalized by the value of the hypoxanthine phosphoribosyltransferase (HPRT) gene.