Ambion dynabeads mrna direct kit

Total RNA extraction and cDNA synthesis were performed as described previously [29 (link)]. Briefly, mRNA was extracted from 50 MII oocytes or 20 blastocysts by means of the Ambion Dynabeads mRNA Direct Kit (Thermo Fisher Scientific). The isolated mRNA was reverse-transcribed with the SuperScript Reverse Transcriptase Kit (LeGene Biosciences, San Diego, CA, USA), using oligo(dT)_12–18 primers, followed by real-time PCR on a Bio-Rad CFX PCR cycler (Hercules, CA, USA) with the primers that were described in our previous paper [26 (link)]. Gene expression was analyzed by the 2^−ΔΔCt method [30 (link)], using data from the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene for normalization. Three separate experiments were carried out, each containing three samples.

Total RNA extraction and cDNA synthesis were performed as described previously [22 (link)]. Briefly, 50 MII-stage oocytes or 20 blastocysts were used to extract mRNA using the Ambion Dynabeads mRNA Direct Kit (Thermo Fisher Scientific). Isolated mRNA was reverse-transcribed with the SuperScript Reverse Transcriptase Kit (LeGene Biosciences, San Diego, CA, USA) using oligo(dT)12–18 primers, followed by real-time PCR in a Bio-Rad CFX PCR cycler (Hercules, CA, USA) with the primers that were described in our previous paper [19 (link)]. Gene expression was analyzed with the 2^-ΔΔCt method [23 (link)], using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a normalizer gene. Three separate experiments were performed, each containing three samples.