Power sybr green pcr master mix

Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed as described previously52 (link)53 (link). Briefly, total RNA was extracted from osteoclasts using TRIZOL (Invitrogen, Carlsbad, CA, USA). First-stranded cDNA was synthesized from 2 μg of total RNA by Easy Script First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China). Quantitative real-time RT-PCR was performed on CFX96 (Bio-Rad, CA, USA) using Power SYBR Green PCR Master Mix (TransGen Biotech, Beijing, China). All reactions were performed in triplicates, and target genes expression was normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used for quantitative real-time RT-PCR are listed in Table 1.

Total RNA was extracted from fresh tissues using TRIzol (Invitrogen, 10296010) and from FFPE tissues using an RNeasy FFPE kit (QIAGEN, 73504) according to the manufacturer's protocol. RNA degradation and contamination were detected on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN). RNA concentration was assessed using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, Q33230). RNA integrity was examined by using an RNA Nano 6000 Assay Kit for the Bioanalyzer 2100 system (Agilent Technologies, 5065‐4401). Further, 2 μg of total RNA was used for PCR, and oligo(dT) was used for cDNA preparation. Real‐Time PCR was performed with Power SYBR Green PCR Master Mix (TransGen) on the ABI 7500 fast real‐time PCR system. The amplification reaction procedure was: 95°C for 10 minutes, followed by 95°C for 15 seconds and 60°C for 1 minute for 40 cycles. GAPDH was used as an internal control, and the relative expression level of mRNA was calculated by relative quantification (2^−△△CT) method. The range of the obtained Ct values was 15‐29. Primer sequences are listed in Table 1.

qRT-PCR was performed as previously described [37 (link), 38 (link)]. Total RNA was isolated with TRIZOL (Invitrogen, Carlsbad, CA, USA) and used for cDNA synthesis with the Easy Script First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. PCR reactions were set up in triplicate using Power SYBR Green PCR Master Mix (TransGen Biotech). Reactions were performed on the CFX96 system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Target gene expression was normalized to the reference gene GAPDH. Primers are listed in Table 1 (annealing temperature = 60°C).

Total cellular RNA was isolated by EasyPure RNA Kit (TransGen Biotech). Total RNA was transcribed to cDNA by a reverse transcription kit (TransGen Biotech). Genomic DNA was extracted and purified from HepG2.2.15 cells by EasyPure Genomic DNA Kit (TransGen Biotech). Real-time PCR was performed with Power SYBR Green PCR Master Mix (TransGen Biotech) in an ABI StepOne Plus Real-Time PCR system (Applied Biosystems). The primer sequence pairs were designed using NCBI online primer blast software. Reactions were performed using 3 µL of cDNA in a 20 µL reaction volume and the following thermal cycle profile: 5 min for pre-denaturation at 94°C, 5 s for denaturation at 94°C, and 30 s for extension at 60°C, for 40 cycles.

Total RNA was extracted using the Trizol reagent (Invitrogen, United States) according to the manufacturer’s instructions. The cDNA was synthesized by using the SuperScript III Reverse Transcriptase Kit (Invitrogen, United States). RT-qPCR was performed with Power SYBR Green PCR Master Mix (TransGen Biotech, China) on the ABI 7500 fast real-time PCR system. The amplification reaction procedure was as follows: 95°C for 10 min, followed by 95°C for 15 s and 60°C for 1 min for 40 cycles. GAPDH was applied as internal control for mRNA, and the relative expression level of mRNA was calculated by relative quantification (2^–ΔΔCT) method. Primer sequences are listed in Table 2.

The total RNA of the cells (HPDE6-C7, Panc-1, Bxpc-3, Aspc-1, and Cfpac-1) was extracted by TRIZOL reagent (TaKaRa, Japan). The RNA samples were reverse transcribed into cDNA using a PerfectStart Uni qPCR + RT kit (AUQ-01, TransGen Biotech Co, Beijing, China). The primer sequences used were as follows: ZBTB4 forward 5′-ACTGTGTCAGATCACTGTGCGAATAG-3′; ZBTB4 reverse 5′-CGTCCTCCTCACTCTCCTCCATC-3′; GAPDH forward 5′-GCACCGTCAAGGCTGAGAAC-3′; and GAPDH reverse 5′-TGGTGAAGACGCCAGTGGA-3′. ZBTB4 expression was determined by quantitative real-time PCR using Power SYBR Green PCR master mix (AUQ-01, TransGen Biotech Co, Beijing, China). Relative mRNA expression levels were calculated by the Ct method, and GAPDH was chosen as the control. Each sample was run in triplicate.

Quantitative reverse transcription PCR (RT-qPCR) was performed by using an Applied Biosystems Step One Plus System and Power SYBR Green PCR Master Mix (TransGen Biotech). RNA was extracted from 50 to 70 oocytes using QIAGEN RNeasy Mini Kit, and cDNA was made by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was treated with RNase H and diluted 1:10 in H₂O, with 8 μl used per PCR. Gapdh was used as a control. Real-time PCR was performed as SYBR Green assays using an ABI 7500 real-time PCR instrument (Applied Biosystems). Primers used in this assay are designed using the software Primer Premier v5.0 (Premier Biosoft International) and shown in Supplementary Table 1. Experiments were performed in biological triplicate and technical duplicate, with data represented as means ± SEM.