Superscript 3 rt first strand synthesis system

Total RNA was isolated from 85 pooled embryos using the RNeasy Micro Kit (QIAGEN, Hilden, Germany; 74004) and from 10 embryos using PicoPure RNA Isolation Kit (Life Technologies; KIT0204). In brief, cDNA was synthesized
from total RNA using Superscript III RT First-Strand Synthesis system (Life Technologies; 18080051). Prepared cDNA samples were amplified and analyzed by reverse transcription-quantitative polymerase chain reaction
(RT-qPCR). The primers used are described in Supplementary Table 1 (online only). Amplified products were run in a 7300 ABI Prism Sequence Detector (Applied
Biosystems). At least three independent experiments were performed for each group.

Total RNA was prepared using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA), and RT was performed using the SuperScript III RT First-Strand Synthesis System according to the manufacturer's instructions (Life Technologies). To confirm PCR amplification, 35 cycles of PCR were performed using a PCR kit (Takara, Kyoto, Japan) on the GeneAmp PCR System 9600 (PE Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C for 10 s, 60°C for 10 s and 72°C for 60 s. An 8-μL aliquot of each reaction mixture was size-fractionated on a 2% agarose gel and visualized using ethidium bromide staining. To confirm RNA quality, PCR amplification was performed for GAPDH using specific primers. For quantitative assessment, gene expression was evaluated by quantitative RT-PCR (qRT-PCR) using the LightCycler CYBR Green Master kit (Roche Diagnostics, Mannheim, Germany) for cDNA amplification of specific target genes. The expression of mRNA copies was normalized against GAPDH mRNA expression. All primer sequences are listed in Table S1.