Dna engine opticon 2 fluorescence detection system

To analyze the expression levels of the genes, mRNA was isolated from denuded MII oocytes, one-cell, two-cell, four-cell parthenotes, morula and blastocyst using the Dynabeads mRNA Direct Kit (Dynal Asa, Oslo, Norway). Ten oocytes/embryos per biological replicate were used for the analysis. First-strand cDNA was synthesized by using Superscript Reverse Transcriptase enzyme (Invitrogen). qRT-PCRs were conducted using DNA Engine Opticon 2 Fluorescence Detection System (MJ Research, Waltham, MA, USA). DyNAmo SYBR Green qPCR Kit (Finnzymes Oy, Espoo, Finland) was used to provide real-time quantification of targeted genes that were amplified with specific primer pairs (Table 1). The PCR was performed as follows: denaturation at 95 8C for 10 min, 40 cycles of amplification and quantification at 94 8C for 10 s, 55 or 60 8C for 30 s and 72 8C for 30 s with a single fluorescence measurement, melting at 65-95 8C with a heating rate of 0.2 8C/s and continuous fluorescence measurement and cooling to 12 8C. The relative quantification of gene expression was determined by the 2 KDDCt method (Livak & Schmittgen 2001) (link) from three technical and biological replicates for both control and CXADR knockdown (KD) group. GAPDH was used as an internal control in all experiments.