Pcr master mix kit

The target genes Cbfα1, MGP and OPN were determined by RT-qPCR, performed using a PCR Master Mix kit (Promega Corporation, Madison, WI, USA) following incubation for 0, 3, 6, 10 and 14 days. The GAPDH gene was used as an endogenous control. The primer sequences used for PCR amplification were as follows: Rat Cbfα1, F 5′-CCG CAC GAC AAC CGC ACC AT-3′ and R 5′-CGC TCC GGC CCA CAA ATC TC-3′ (generating an amplified fragment of 289 bp); rat MGP, F 5′-AAA GCC CAG GAA AGA GTC CG-3′ and R 5′-TCT TAT TTG GCT CCT CGG CG-3′ (generating an amplified fragment of 158 bp); rat OPN, F 5′-ATG CTA TCG ACA GTC AGG CG-3′ and R 5′-GCT CAG GGC CCA AAA CAC TA-3′ (generating an amplified fragment of 317 bp); rat GAPDH, F 5′-CCC ACT AAA GGG CAT CCT GG-3′ and R 5′-GGC CCC TCC TGT TGT TAT GG-3′ (generating an amplified fragment of 352 bp). The PCR products (5 μl) were subjected to electrophoresis (MultiSUB Midi-96; Beijing Thmorgan Biotechnology Co., Ltd., Beijing, China) using a 1.5% agarose gel and visualized with an ethidium bromide stain (all from Invitrogen Life Technologies, Carlsbad, CA, USA). The band optical density was measured using a Gel Documentation System (CST Biological Reagents Company Limited, Shanghai, China) and the final data are expressed as the mRNA level relative to that of GAPDH.

The miR-SNP of the miRNA processing genes including XPO5 (rs11077), RAN (rs14035), Dicer(rs3742330), TNRC6B(rs9623117), GEMIN3(rs197412), GEMIN4(rs2740348) were genotyped using the Polymerase Chain Reaction - Ligase Detection Reaction (PCR-LDR) assay with the forward and reverse primers to amplify the DNA fragments flanking miR-SNPs using the sequence in the NCBI SNP database (http://www.ncbi.nlm.nih.gov/snp/). Polymerase Chain Reaction (PCR) was performed using a PCR Master Mix Kit according to the manufacturer's instructions (Promega, Madison, WI). The ligation was performed using the different probes matched to the miR-SNPs, and the ligated products were separated using the ABI PRISM Genetic Analyzer 3730XL (Applied Biosystems, Foster City, CA). Polymorphisms were confirmed based on the length difference of ligated products. All the primers and probes were listed in Table 1.

Genomic DNA was used to amplify the fungal ITS region (ITS1-5.8S-ITS2) applying the primer pairs ITS4 and ITS5 [29 ]. For amplification reactions, a PCR Master Mix Kit was used (Promega, Madison, WI, USA), following the manufacturer’s instructions. To visualize the amplification, product electrophoresis was performed on a 1% agarose gel stained with Nancy dye (Sigma–Aldrich, Saint Louis, MO, USA). Next, the amplification products were purified using the Wizard^® SV Gel kit and PCR Clean-Up System (Promega) following the kit’s instructions. Finally, the PCR product was quantified on a NanoDrop^® (Thermo Scientific, Waltham, MA, USA).

Total cellular RNA was isolated using a RNeasy Mini Kit (Qiagen, Valencia, CA; https://www.qiagen.com/) and treated with a DNA-free kit (Ambion, Austin, TX; http://www.lifetechnologies.com/) to remove potential contamination of genomic DNA. A total of 500 ng of RNA was used as a template for reverse transcription with Reverse Transcription System (Promega, Madison, WI; http://www.promega.com/). 100 ng of cDNA was used for a standard PCR reaction. A housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a control for the PCR efficiency of each sample. The PCR step was performed using PCR Master Mix kit (Promega, Madison, WI), and the PCR products were detected and analyzed by 2% agarose gel electrophoresis. 293 T cells were as negative control.
CXCR4 primers were as follows: forward 5′-ATG GAG GGG ATC AGT ATA TAC AC-3′ and reverse 5′-TGG AGT GTG CTA TGT TGG CGT CT-3′ (product 668 bp); GAPDH primers were forward 5′-ACC-ACA-GTC-CAT-GCC-ATC-AC-3′ and reverse 5′-TCC-ACC-ACC-CTG-TTG-CTG-TA-3′ (product 450 bp).

Total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with primers for human ATF3 and human GAPDH as follows: human ATF3: 5′-gtttgaggattttgctaacctgac-3′, and reverse 5′-agctgcaatcttatttctttctcgt-3′; huaman GAPDH: forward 5′-acccagaagactgtggatgg-3′ and reverse 5′-ttctagacggcaggtcaggt-3′.

After treatment, total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with human primers for cyclin D1 and GAPDH as followed: cyclin D1: forward 5′-aactacctggaccgcttcct-3′ and reverse 5′-ccacttgagcttgttcacca-3′, GAPDH: forward 5′-acccagaagactgtggatgg-3′ and reverse 5′-ttctagacggcaggtcaggt-3′. The following PCR reaction conditions were used: 1 cycle of (3 min at 94 °C for denaturation), 25 cycles of (30 s at 94 °C for denaturation, 30 s at 60 °C for annealing, and 30 s at 72 °C for elongation), and 1 cycle of (5 min for extension at 72 °C).