Primetime predesigned qpcr assay 6 fam zen ibfq

Total RNA was isolated using Quick-RNA™ MicroPrep (Zymo Research) following the manufacturer’s instructions and was reverse-transcribed on a GeneAmp® PCR System 9700 (Applied Biosystems). Real-time quantitative PCR reactions were performed on a 7900 HT FAST Realtime PCR System (Applied Biosystems). The cDNA equivalent of ~ 5 embryos per stage was used for each target gene. PrimeTime®Predesigned qPCR Assay (6-FAM/ZEN/IBFQ) from Integrated DNA Technologies was used. Assay IDs: Trim21: Mm.PT.5812570300 and β-Actin: Mm.PT.39a.22214843.g. All samples were processed as technical duplicates. Data were analyzed with the ΔΔCt method [73 (link)] using the Applied Biosystems RQ Manager (Version 1.2.2) and Microsoft Excel. ∆∆Ct = ∆ (Ct Trim21 - Ct β-Actin of mCherry-Trim21 injected embryos) – ∆ (Ct Trim21 - Ct β-Actin of non-injected wt embryos). Ct: cycle threshold.

For each target gene, the cDNA equivalent of 10 MII oocytes or embryos per stage was used. Total RNA was isolated from large pools (>100 oocytes or embryos) using Quick-RNA™ MicroPrep (Zymo Research) following the manufacturer’s instructions and was reverse-transcribed on a GeneAmp® PCR System 9700 (Applied Biosystems). Real-time quantitative PCR reactions were performed on cDNA on a 7900 HT FAST Realtime PCR System (Applied Biosystems). PrimeTime®Predesigned qPCR Assay (6-FAM/ZEN/IBFQ) from Integrated DNA Technologies were used. Assay IDs: Dnajb11_Mm.PT.58.9272431, Pdia3_Mm.PT.8194853; Top1_Mm.PT.58.6752545. All samples were processed as technical duplicates/replicates. Data were analyzed using the Applied Biosystems RQ Manager (Version 1.2.2) and Microsoft Excel.