Icycler iq5tm real time pcr detection system

First strand cDNA synthesis was done using iScript™ cDNA synthesis kit (Biorad #170-8897) by following manufacturer’s protocols. The cDNA obtained was used to perform real-time qRT-PCR in a Bio-Rad iCycler iQ5TM Real-Time PCR detection system using 2X iQ SYBR Super mix (Invitrogen #K0242). The sequences of primers (from 5′ end to 3′ end) used for qRT-PCR are followed. CyclinD1-F′-TCCAGAGTGATCAAGTGTGA-3′, CyclinD1-R-GATGTCCACGTCCCGCACGT, CDK1-F-CCTTGCCAGAGCTTTTGGAATACC-3′,CDK1-R-GACATGGGATGCTAGGCTTCCTGG, cyclinB-F-TGGAAAAGTTGGCTCCAAAG, cyclin B-R-TCAGAAAAAGCTTGGCAGAGA, Actin-F-GTGGGCATGGGTCAGAAG, Actin-R-TCCATCACGATGCCAGTG. The gene expression levels were determined using the cycle threshold (C_t) method. The mean C_t values from duplicate measurements were used to calculate the expression of the target gene with normalization to a housekeeping gene and the difference in fold expression was calculated using ΔΔC_t method.

The first-strand cDNA synthesis was done with the iScriptTM cDNA synthesis kit (Biorad) per the manufacturer’s instructions. The obtained cDNA was used to carry out RT-qPCR (Bio-Rad iCycler iQ5TM Real-Time PCR detection system) using 2X iQ SYBR Supermix (Invitrogen). The primer sequences (from 5ʹ end to 3ʹ end) used for qRT-PCR are as follows:
TREX1-F-GAGAGTGTGCAGCCGAGTCA, R-AGATGAGGGTCTGCATGGGC.
Β-Actin-F-GTGGGCA TGGGTCAGAAG, β-Actin-R-TCCATCACGATGCCAGTG.
The levels of gene expression were assessed by the cycle threshold (Ct) method. Mean values of Ct from duplicate results were used to calculate the target gene expression with housekeeping gene normalization. The difference in the fold expression was finally calculated through the ΔΔC_t method.