Iq5 pcr system

Kaempferol (1,000 ng/ml; Sigma) was used to stimulate C2C12 cells for 24 h before harvest. TRIzol (Invitrogen Life Technologies, Carlsbad, CA, United States) was used to extract total RNA from cultured cells as previously described (Tang et al., 2012 (link)). RNA purity and integrity were determined by the RNA 6000 Nano assay with an Agilent Bioanalyzer 2,100 (Agilent Technologies, Santa Clara, CA). 1 μg total RNA was reverse transcribed into cDNA using the GoScript Reverse Transcription System (Promega Corp., Madison, WI, United States) based on the manufacturer protocol. qPCR was performed in triplicate using SYBR-Green SuperReal PreMix Plus [Tiangen Biotech (Beijing) Co., Ltd., China] and the iQ5 PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, United States). The reaction conditions were as follows: 95°C for 30 s, and 40 cycles of 95°C for 10 s and 60°C for 30 s. Data were reported as cycle threshold (Ct) values. The 2^−ΔΔCt method was used to compare the RNA expressions. RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.

rBMSCs and ROB were harvested and lysed at 0 h, 6 h, 12 h, 24 h, and 48 h after the osteogenic induction culture and were subjected to qRT-PCR analyses to evaluate the effects of 7-methoxycoumarin and osthole on mRNA expression of the osteogenesis-related genes, including insulin-like growth factor-1 (IGF-1), Osterix, Runx-2, OPG, and RANKL, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control following the protocol described previously [23 (link)]. Total RNA was extracted by using RNAprep pure Cell Kit and treated with DNase I to remove any DNA contamination. The concentration and purity of total RNA were determined by absorbance at 260/280 nm in a UV spectrophotometer, and the integrity was checked by 1.5% agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized by using Quantscipt RT Kit in 20 μL reactions containing 1 μg total RNA. qRT-PCR reaction was carried out in iQ5 PCR system (Bio-Rad, USA) by using RealMasterMix PCR Kit. Primers were designed with the Primer Express 3.0 software based on published rat cDNA sequences in NCBI and the sequences were listed in Table 1.

The mRNA expressions of BDNF, TrkB, and p75NTR were measured by quantitative real-time PCR. Total RNA was isolated from the brain tissues using an RNeasy kit with Trizol (Invitrogen, USA), and reverse-transcribed into cDNA using a reverse transcription kit with M-MLV polymerase (Promega, USA). The sequence-specific primers used were as follows: BDNF-F, 5′-CGATTAGGTGGCTTCATAGGAG-3′ and BDNF-R, 5′-ACGAACAGAAACAGAGGAGAGATT-3′; TrkB-F, 5′-CAAGTTGGCGAGACATTCCA-3′ and TrkB-R, 5′-AGTCATCGTCGTTGCTGATGAC-3′; p75NTR-F, 5′-TTCCTTAGCCCCTCCCTTCT-3′ and p75NTR-R, 5′-CCTGCCTTTCTCTGGGTTTTAC-3′; ACTIN-F, 5′-GCTATGTTGCCCTAGACTTCGA-3′ and ACTIN-R, 5′-GATGCCACAGGATTCCATACC-3′. PCR was performed using an IQ5 PCR System (Bio-Rad, USA) with an initial denaturing step at 95 °C for 15 s, 45 cycles of denaturing at 95 °C for 5 s, and annealing at 60 °C for 30 s. The relative expressions of genes were determined using the ΔΔCt method [22 (link)] to normalize the target gene expression to that of the housekeeping gene (ACTIN).

Total traumatized cutaneous-tissue RNA was extracted using an RNA Fast Kit (Yeasen Biotechnology, Shanghai, China). We synthesized cDNA using a HiScriptII Reverse Kit (Roche). Real-time polymerase chain reaction (RT-qPCR) was performed using FastStart Universal SYBR Green PCR Mix on an IQ5 PCR System (BioRad Laboratories, Hercules, CA, United States). The reaction procedure was as follows: 40 cycles of 94°C for 1 min, 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s. Primers used are listed in Table 1. We calculated mRNA expression of related target genes using the classic formula 2^−xpre. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal gene.

Total RNA was extracted by using a RNA Fast kit (Solarbio, China). A HiScript®II Reverse Kit (Vazyme, China) was used to perform cDNA synthesis. The qPCR assay was performed by using SYBR Green PCR Mix (Boster, China) on an IQ5 PCR System (Bio-Rad, USA). The reaction procedures were as follows: 94°C for 1 min, 94°C for 30 s, 55°C for30 s, and 72°C for 30 s for 40 cycles. The following primers were used: circ-GTF2I, forward: 5′-ACAGAATTCGCCACCATGCTGGCCGTCGGCTG-3′, reverse: 5′- ACAGGATCCTCTGGGGAAGAAGTAGTCTGTATTGCTGAT-3′, Bax, forward: 5′-CTTCCAAGCCCACCCCAACT-3′, reverse: 5′- GGCCTCCAGGACCTTCAGC-3′, Cyt-c, forward: 5′-CTCCTCTGCATTGCCATTGT-3′, reverse: 5′-TGTGGCTCGAGGTATTGTCA-3′, Bcl-2, forward: 5′-CGACAAGCCTCCCAGGTTCA-3′, reverse: 5′- GTGCCACCCAGCCAGCTATC-3′, KBTBD7, forward: 5′-AAAGGAAGGGGACTACCAAAGAAA-3′, reverse: 5′-CACCTCCTCCACATCATACACCTG-3′, miR-590-5p, forward: 5′-TAGCTTATCAGACTGATGTTG-3′, reverse: 5′-TCAACATCAGTCTGATAAGCT-3′, U6, forward: 5′-GCTTCGGCAGCAACATATAC-3′, reverse: 5′-AACGCTTCACGAATTTGCGT-3′, GAPDH, forward: 5′-TGAGACCTTCAACACCCCAG-3′, reverse: 5′- GCCATCTCTTGCTCGAAGTC-3′.
Quantitative analysis was performed by comparing 2^−ΔΔCt values. The analysis was repeated 3 times for each set. The expression of KBTBD7 mRNA was corrected on the basis of GAPDH expression. U6 was used to correct miRNA expression results.