One step rt pcr kit

The total RNA of cell lines and tissues were extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and were reverse transcribed to cDNA via one-step RT-PCR kit (TransGen Biotech Co., Ltd, Beijing, China) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction was performed in an Applied Biosystems 7500 Detection system with SYBR Green (TransGen Biotech Co., Ltd, Beijing, China). The relative mRNA levels of genes were normalized to peptidylprolyl isomerase A (PPIA) [32 (link)] using the 2^-ΔΔCT method. The primer sequences are given in Additional file 1: Table S2.

The one-step RT-PCR kit (TransGen Biotech Co., Ltd., China) was used to assess the reverse transcription of total RNA. The primers used for polymerase chain reaction were as follows: α-SMA, forward—5′-AAGTATCCGATAGAACAC-3′, reverse—5′-AAACATAATCTGGGTCAT-3′; Runx2, forward—5′-AGGCGCATTTCAGATGATGAC-3′, reverse—5′-ACCTGCCTGGCTCTTCTTAC-3′; β-actin, forward—5′-GATGGTGGGTATGGGTCAGAAGGAC-3′, reverse—5′-GCTCATTGCCGATAGTGATGACT-3′. The ABI PRISM 7500 Sequence Detection System using QuantiTect SYBR Green (QIAGEN, Hilden, Germany) was used to quantify differential gene expression. The mRNA expression levels were normalized to β-actin mRNA.

Total RNA was extracted from dissected hippocampal samples on the 14th day after exposure using TRIzol reagent (Invitrogen, USA). RNA levels were quantified using a Nanodrop 1000 spectrometer (Thermo Scientific, UK). Reverse transcription of mRNA was performed using a First-Strand cDNA Kit (AT311, TransGen, China) and a One-Step RT-PCR Kit (AQ131, TransGen, China) with SYBR Green (TransGen, China) according to the manufacturer's instructions. Amplification was conducted using a 7500 real-time PCR system (Applied Biosystems, USA). GAPDH, a housekeeping gene, was used as an internal control to standardize 5-HT_1A receptor mRNA expression. Relative expression (R) of the 5-HT_1A receptor gene was calculated using the 2^−ΔCT method (ΔCT = CT_(5−HT1AR) - CT_(GAPDH)). The primers were designed using Primer Premier 5.0 software. The primer sequences were as follows: 5-HT_1A receptor forward, 5′-AGTGAGGCAGGGTGACGACG-3′, and reverse, 5′-CAGGACCAGAGCCACAATGAAA-3′ (265 bp); GAPDH forward, 5′-GATTTGGCCGTATCGGAC-3′, and reverse 5′-GAAGACGCCAGTAGACTC-3′ (278 bp).

Total RNA was extracted from the cells using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and was reverse transcribed into a total of 1 μl (60 ng/μl) cDNA using a One-Step RT-PCR kit (TransGen Biotech Co., Ltd., Beijing, China), according to the manufacturer’s instructions. Quantification of gene expression was performed using an ABI PRISM 7500 Sequence Detection system (Applied Biosystems Life Technologies, Foster City, CA) with SYBR® Green (TransGen Biotech Co., Ltd.). The relative mRNA expression levels of SDF-1α and CXCR4 were normalized to that of β-actin using the 2^−ΔΔCT method (20 (link)). The following primer sequences were used (Shanghai GenePharma Co., Ltd., Shanghai, China): Rat CXCR4, forward 5′-GCTGAGGAGCATGACAGACA-3′ and reverse 5′-GAT GAAGGCCAGGATGAGAA-3′ (21 (link)); rat SDF-1α, forward 5′-CTGTTGTGCTTACTTGTTTAAGGCTTTGTC-3′ and reverse 5′-GACGCCAAGGTCGTCGGT-3′ (22 (link)) and rat β-actin, forward 5′-GCTACAGCTTCACCACCACA-3′ and reverse 5′-GCCATCTCTTGCTCGAAGTC-3′. The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 72°C for 35 sec, for telomere PCR. The experiments were repeated three times with triplicates of each sample.

HCV antibody testing was conducted using enzyme-linked immunosorbant assay (ELISA) with HCV-IgG ELISA kit (Wantai, Beijing, China), and positive results were repeated twice for accuracy. HCV RNA was quantified by real-time PCR, using Roche light cycle 2.0 Diagnostic Kit for hepatitis C virus RNA (PCR-Fluorescence Probing) (Da’an, Shenzhen, China). HCV RNA positive was defined as RNA copies over 0.5 × 10³ IU/ml. Samples with enough leftovers were further sent for nested PCR and sequencing. Nested PCR was performed as follows: the first round RT-PCR was performed using One-Step RT-PCR kit (TransGene Biotech, Beijing, China) using NS5B and Core specific external primers with the following protocol: 45 °C 30 min, 94 °C 2 min, (94 °C 30s, 50 °C 30s, 72 °C 90s) × 35 cycles, and 72 °C 10 min; the second round PCR was performed using Promega Taq PCR Mastermix kit (Promega Company, USA), using 5ul product from the first round as the template, and NS5B and Core specific internal primers (Additional file 1: Supplementary Information 3 and Table S2), with the following protocol: (94 °C 2 min, 58 °C 30s, 72 °C 90s) × 30 cycles, and 72 °C 10 min. The final PCR product was tested in agar gel electrophoresis and sent for Sanger sequencing.