Cfx connect real time pcr detection system machine

Total RNA was extracted from testes dissected from one-day-old males with TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) and a Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA, USA) with DNase. cDNA was synthesized with random hexamer oligonucleotides and SuperScript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Quantitative real-time polymerase chain reaction (qPCR) assays were conducted with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) on a CFX Connect Real-Time PCR Detection System machine (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s suggested parameters. Measurements were made on three biological replicates for Oregon R, Z3-5009 and bamGal4 > Pif1ARNAi and one replicate for Pif1A-GFP; Z3-5009 and technical triplicates for each sample. The housekeeping gene ribosomal protein 49 (RpL32) was used as a control to normalize mRNA expression. Data were transformed and analyzed according to the ΔΔCt method with Bio-Rad CFX Manager software 3.0 (Bio-Rad Laboratories, Hercules, CA, USA). The following primers were used for qPCR: Pif1A-RI-forward: 5′-ATGAGGTACCGAGCCTCCA-3′; Pif1A-RI-reverse: 5′-GGGCTCTTTGCTTTGGAGA-3′; rp49-forward: 5′-CACCAAGCACTTCATCCG-3′; rp49-reverse: 5′-TCGATCCGTAACCG ATGT-3′.

Total RNA was extracted from FLS using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. Extracted RNA was reverse transcribed with ReverTra Ace^® qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. SYBR^® Green Real-Time PCR Master Mix (TOYOBO) was used for qRT-PCR analysis of cDNA according to the manufacturer’s instructions. Thermal cycling was conducted on a CFX Connect Real-Time PCR Detection System machine (Bio-Rad Laboratories, Hercules, CA, USA). Target gene expression levels are shown as a ratio of the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the same sample via calculation of the cycle threshold (Ct) value. Relative expression levels of target genes were calculated using the 2^−ΔΔCT relative quantification method.
For RT-PCR, the synthesized cDNA was mixed with AccuPower^® RT PreMix (Bioneer, Daejeon, Republic of Korea; see Table 2 for primer sequences) and specific PCR primers following the manufacturer’s protocol. Amplified products were separated on 1% agarose gels, stained with Midori green advance (NIPPON Genetics, Düren, Germany), and photographed under UV illumination using a GelDoc system (Bio-Rad Laboratories).