Transcript nova kit

The total RNA was manually isolated from the cumulus cells using Trizol reagent (Sigma Pool, UK) following the manufacturer’s protocol. The transcript nova kit (Qiagen Inc., Valencia, CA, USA) was used for reverse-transcription of obtained RNA into cDNA as a described previously [20 (link)]. The reverse-transcribed yields of Ahr, Arnt, Cyp1A1, and Cyp1B1, were amplified by real-time polymerase chain reaction (PCR) with SYBR Green (Takara, Japan) on an ABI real-time PCR system (Applied Biosystems, ABI, Foster City, CA, USA), according to the manufacturer’s instructions. Finally, all data were analyzed by the standard formula, while glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal reference gene. Primer sequences were as follows: human AHR (sense, 5´-AGAGTTGGACCGTTTGGCTA-3´; antisense, 5´AGTTATCCTGGCCTCCGTTT-3´), human ARNT (sense, 5´- CAAGCCCCTTGAGAAGTCAG-3´; antisense, 5´-GGGGTAGGAGG GAATGTGTT-3´), human CYP1A1 (sense, 5′-TCA ATC AAG AGG CGC GAA CCT C-3′; antisense, 5′-CTA CAG CCT ACC AGG ACT CG-3′, human CYP1B1 (sense, 5´-AAGTTCTTGAGGC ACTGCGAA-3´; antisense, 5´-GGCCGGTACGTTCTCCAAAT-3´), and human GAPDH (sense, 5´-TGGACCTGACCTGCCGTCTA-3´; antisense, 5´-CTGCTTCACCACCTTCTTGA-30).

After denuding and washing of mature oocytes, 10-15 oocytes were accumulated four times, and total RNA was isolated from these accumulations. The RNeasy Micro kits (Qiagen Inc., Valencia, CA, USA) and transcript nova kit (Qiagen Inc., Valencia, CA, USA) were used for RNA isolation and reverse-transcription into cDNA as a described previously (20). The reverse-transcribed yields of two subunits of maturation-promoting factor (MPF: Cdk1, cyclin B), mitogen-activated protein kinase (Mapk), Cyclooxygenase (Cox2), Glutathione peroxidase (Gpx1), DNA methyltransferase-1 (Dnmt1) and histone deacethylase-1 (Hdac1) were amplified by real-time polymerase chain reaction (PCR) with SYBR Green (Takara, Japan) on an ABI real-time PCR system (Applied Biosystems, ABI, Foster City, CA, USA), according to the manufacturer's instructions. Finally, all data were analyzed by the standard formula, while glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal reference gene. The details of real-time PCR reaction was described in our previous article (17). Table I lists the primer sequences.