Abi 7700 system

Total cellular RNA was extracted using TRIzol reagent (Invitrogen) and 5 μg of extracted RNA samples reversely transcribed into cDNA according to the manufacturer's protocol (Superscript III, Invitrogen). The RT-qPCR was performed using the SYBR Green Mix containing Thermo-Start DNA polymerase (Bio-Rad, Hercules, CA) according to the manufacturer's instructions, as regards the ABI7700 System (Applied Biosystems, Foster City, CA), with specific primers used (Supplementary Data Table 2).

Total RNA was extracted via the SV total RNA isolation kit (Promega), and reverse transcription was performed through the TOYOBO ReverTra Ace-a RT-PCR kit (TOYOBO, Japan) [12 (link)]. cDNA was mixed with SYBR Green PCR Master Mix. Quantitative real-time PCR (qRT-PCR) was analyzed through the ABI-7700 system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers, forward, 5′-GAAGG TGAAGGTCGGAGTC-3′; reverse, 5′-GAAGATGGTGA TGGGATTTC-3′ [12 (link)]. Gab2 primers, forward, 5′-CGAA GAGAACTATGTCCCTATGC-3′; reverse, 5′-AGGGGCA GGACTGTTCGT-3′ [36 (link)]. After amplification, melt curve analysis was performed to calculate product melting temperature. For normalization, GAPDH was utilized as the reference gene, and ^ΔΔCt method was applied. The detection of mature miR-302c-3p was through the TaqMan microRNA assay of has-miR-302c-3p (Applied Biosystems, Shanghai, China) (see method in [37 (link)]). Twenty ng of RNA was reverse-transcribed by the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) and the looped primer provided by the specific TaqMan microRNA assay.

qRT-PCR for target genes was performed by using a SYBR Green kit (Applied Biosystems, Foster City, CA, USA). PCR was carried out on an ABI 7700 system (Applied Biosystems, Foster City, CA, USA) using the following reaction conditions: 15 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 64 °C. All gene expression levels were normalized to CypA (cytochrome P450 A) expression. The primers are listed in Table 1.

RNA was isolated as previously described. Reverse transcription was performed by using a miScript^TM reverse transcription kit (Qiagen, Hilden, Germany). For miRNA expression, qRT-PCR was performed by using a TaqMan PCR kit (Applied Biosystems, Foster City, CA, USA) and an ABI 7700 system (Applied Biosystems, Foster City, CA, USA). The reaction conditions were as follows: 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, and 60 s at 60 °C. miRNA expression levels were normalized to U6 expression.

All primers in this study were chosen using a sequence analysis software package (Primer 3, Whitehead Institute for Biomedical Research, Cambridge, MA) following guidelines for internal stability. Forward and reverse primers were in different exons to minimize the effects of possible DNA contamination. The primers for genes of 3β-HSD1 (Hsd3b1) and 11β-HSD1 (Hsd11b1) and 11β-HSD2 (Hsd11b2) were reported in the previous study [18 (link)]. For the internal standard, primers to ribosomal protein S16 (Rps16) were as described previously [18 (link)]. The primers for H6PDH (H6pd) and G6P transporter (Slc37a4) were (forward and reverse pairs): 5'-ACCGGGGACCTAGCTAAAAA-3', 5’-CTCAAGATTATCCCAGAGG-3’ for H6pd and 5’-TTGGACTCCGAAATCTGGAC-3’, 5’-GGTCACTGTTACCCGGAAGA-3’ for Slc37a4. First strand cDNAs, synthesized using total RNA from adult rat testis as described previously [18 (link)], were used as templates for PCR. Real-time PCR (qPCR) was carried out in a 25-μl volume using a 96-well plate format using the SYBR Green PCR Core Reagents purchased from Applied Biosystems (Foster City, CA) by a standard curve method as described previously [18 (link)]. Primer titration was performed and the concentration of 300 nM was selected. Fluorescence was detected on an ABI 7700 system (PE Applied Biosystems). Each sample was run in duplicate. The levels were normalized to Rps16.

Briefly, first‐strand synthesis of DNA and RT‐qPCR were performed as previously described.³⁸ qPCR was carried out in a 20 µl volume in a 96‐well plate using the SYBR Green PCR Kit from Applied Biosystems. Primer titration was performed with the concentration of 300 nmol/L. Fluorescence was detected using the ABI 7700 system (PE Applied Biosystems). Each sample was run in duplicate and in parallel with no template controls. The relative mRNA levels of targeted genes were adjusted to housekeeping gene, ribosomal protein S16 (Rps16), as the internal control. Rsp16 in LCs has been used as the internal control in many studies because it showed consistent expression.³², ³⁹ The Ct value was read, and the levels of the target mRNAs were calculated using the standard curve method as previously described.³⁶ All primers in the present study were designed by Primer 3 software (Whitehead Institute for Biomedical Research). Forward and reverse primers were placed in different exons to minimize the effects of possible DNA contamination. These genes are as follows: luteinizing hormone receptor (Lhcgr), scavenger receptor class B member 1 (Scarb1), Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, Akr1c9, insulin‐like 3 (Insl3), sulfotransferase 1A1 (Sult1a1) and cyclin D1 (Ccnd1). The primers were listed in Table S1.