7500 real time pcr system

Real-time quantitative reverse transcription-PCR was performed either with a Biorad MyiQ™ Single-Color Real-time PCR System (at SUNY Buffalo) or an ABI 7500 Real-time PCR System (at UTMDACC). The cDNAs were synthesized from 1.5 ìg of total RNA using SuperScript III reverse transcriptase (200 U/ìl) (Invitrogen) with random hexamers. The gene-specific primers are summarized in Table S1 in File S1. The reaction components were added as follows: 5 µl forward primer (5 µM), 5 µl reverse primer (5 µM), 12.5 µl SYBR Green (Applied Biosystems), 1 µl cDNA, Taq polymerase, and water to 25 µl. The cycling profile for each run consisted of 50°C for 3 min, template denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, a primer annealing-elongation step at 60°C for 30 s using the default ramp rate. This was followed by a final step of 40°C for 1 min. All reactions were run in triplicate and the mean values were used in the analysis. The cycle threshold (C_t) values were determined using the instrument software. The change in gene expression levels was determined by normalizing mRNA levels of the gene of interest to the mRNA level of the housekeeping gene glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) using the comparative C_t method.

Cells of PD1222, PD-pBBR and PD-pdeR were harvested when OD₆₀₀ was about 1.5. Total RNAs were isolated and purified with an RNeasy mini system (Qiagen, Germany) and then reverse transcribed by PrimeScript^™RT Master Mix (Takara, Japan) according to manufacturer’s instructions. Quantitative PCR reactions were performed using the SYBR^® Premix Ex Taq Kit (Takara, Japan) and a 7,500 real-time PCR system (Bio-Rad, United States). The primers used in Real-time Quantitative PCR (RT-qPCR) analyses were designed by Primer premier 5 and are listed in Table 2. The relative expression of pdeR was calculated by the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)) with 16 s rRNA of P. denitrificans used as an internal control. All reactions were carried out in triplicate.

The primers for qRT-PCR (Table S2) were designed by Oligo and synthesized by Sangon Biotech (Shanghai, China). Total RNA was extracted using TRIzol (Solarbio, Beijing Science & Technology Co. Ltd., Beijing, China) and its purity was measured by NanDrop 2000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Then, a reverse transcription kit of Strand cDNA Synthesis SuperMix for qPCR (Yeasen Biotech Co. Ltd., Shanghai, China) was used to synthesize cDNA from RNA. Using qPCR SYBR Green Master Mix (Yeasen, Shanghai, China), qRT-PCR was performed using a 7500 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). PCR amplification program was shown in Table S3. The fold change of target gene expression was analyzed according to the formula proposed by Pfaffl: 2^−ΔΔCt.

TRIzol reagent (Invitrogen, Carlsbad, United States) was used to extract the total RNA from the primary granulosa cells according to the manufacturer’s protocol, and the RNA concentration was measured by NanoDrop (Thermo Fisher Scientific). RNA was reverse transcribed into cDNA using the RT Kit (Bio-Rad Laboratories, Hercules, United States), and real-time PCR was performed on a 7500 Real-Time PCR System (Bio-Rad Laboratories) using 5 × SYBR Green SuperMix (Bio-Rad Laboratories). The method of 2^–△△Ct was used to calculate the relative expression of the target gene as compared to that of the internal reference: △Ct = △Ct_target – △Ct_reference, – △△Ct = – (△Ct_treat – △Ct_control). The sequences of the primers (Invitrogen) are shown in the Supplementary Table 1. The melting curve was used to verify the primer specificity. GAPDH was used as an internal reference gene.

According to the mRNA sequence of chicken Bax, Bcl-2, Cyt C, Caspase 3, Caspase 9 and β-actin in GenBank, the primers for QRT-PCR were designed by Oligo and synthesized by Beijing Genomics Institution (shown in Table 1). A total of 25–30 mg of liver was used to make tissue homogenate. RNA was extracted by TRIzol (Vazyme Biotech Co, Nanjing, China), and converted to cDNA via a reverse transcription kit HiScript Q RT SuperMix (Yeasen Biotech Co, Shanghai, China) for qPCR. Using the SYBR Premix Ex Taq TM II kit, qRT-PCR was performed with the 7500 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). According to the mathematical model proposed by Pfaffl, the fold change of target gene expression was calculated using the formula: 2^−∆∆Ct.

Total RNA was extracted from tubers using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA was obtained with a reverse transcriptase kit (Thermo, Tokyo, Japan). The qRT–PCR assay was performed following the manufacturer’s protocol using a 7500 Real Time PCR System (Bio-Rad, CA, USA). The relative gene expression levels were calculated using the formula 2^-ΔΔCt. Elongation factor 1 alpha-like (EF1αL) was defined as the internal reference gene, and the primer sequences are provided in Supplementary Table S2.