Quantinova sybr green pcr kit

A subsample of collected swabs was tested in duplicate for total DNA content using the Quant-iT™ dsDNA Assay Kit (#Q33120) (ThermoFisher Scientific, Australia). qPCR was performed on all swab samples using bacteria-specific SSU rRNA primers (1406F/1525R). The PCR was prepared using 5 µL of 2X QuantiNova SYBR Green PCR Kit (Qiagen, Germany), 4 µL of skin swab DNA and 1 µL of primer mix. Samples were run in triplicate on the ViiA7 platform (Applied Biosystems, USA). Cycling conditions and preparation of the quantification standard are described in the supplements. Bacterial SSU rRNA gene copy numbers were calculated using QuantStudio Real-Time PCR Software v1.3.

RT-qPCR used B. subtilis 16S ribosomal RNA as the internal control. Total RNA was extracted from each sample using RNA fast200 Kit (FASTAGEN, Shanghai, China, Cat#220010 50), according to the manufacture’s instruction. Then, 5 μg of total RNA was reverse transcribed with qPCR RT Master Mix Kit (TOYOBO, Osaka, Japan, Cat#FSQ-301) for complementary DNA synthesis. RT- qPCR was performed in a 20 μL volume consisting of 10 μL 2X QuantiNova SYBR Green PCR Kit (QIAGEN, Hilden, Germany, Cat#208054), 8 μL RNA-free water, 1 μL cDNA template, and 0.5 μL of each primer pair according to the manufacturer’s procedure using CFX Connect Real-Time PCR System (BIO-RAD, Hercules, CA, USA). Three biological replicates were performed each with three technical replications. Relative differential expression levels of msyB were calculated based on the cycle threshold (C_T) values normalized with the control gene using the 2^−△△CT method.

Total mRNA was extracted from wound tissue (60 mg) using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). Trizol^® (Invitrogen Corporation, CA, USA) was used for RNA extraction from cultured neutrophils and the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) was used for cDNA synthesis. Reactions were performed using QuantiNova SYBR-Green PCR Kit (Quiagen^®) or iTaq Universal SYBR Green Supermix (1725121, Bio-Rad^®) in a StepOnePlus™ RT-qPCR System. Ubiquitin C (UBC) and beta-2-microglobulin (B2M) were used as endogenous controls. The delta-delta cycle threshold (ΔΔCt) method was used to calculate fold changes (31 (link)). Supplementary Table 1 shows the primer sequences used for the RT-qPCR experiments.

Total RNA was isolated using the RNeasy Kit (Quiagen). A total of 2 μg of RNA were used for cDNA synthesis using the Maxima First Strand cDNA Synthesis Kit (Thermo Fischer Scientific) according to the manufacturer's instructions. qRT-PCR was performed with gene- and species-specific primers. cDNA template was amplified using the QuantiNova SYBR GreenPCR Kit (Quiagen) on a Rotor-Gene (Quiagen) according to the manufacture’s protocol. Quantitative analysis of data were performed by using the deltadeltaCT method.

mRNA from 10⁶ cells was extracted from cells on the differentiation phase using the RNeasy Mini Kit (Quiagen, Hilden, Germany) as recommended by supplier. 1 μg of RNA was used for cDNA generation using a High-Capacity Reverse Transcription Kit (Life Technologies) as recommended by supplier. Quantitative real-time PCR (RT-qPCR) was performed on 7300 Real Time PCR system (Life Technologies) using a Quantinova SYBR Green PCR Kit (Quiagen) and analysed with the software provided by supplier. RT-qPCR primers were designed using Primer3 software and are listed on Supplementary Table 4. For each primer pair, amplification specificity was validated by melting curve and gel electrophoresis. Relative expression was normalized to unvarying polyadenylate-binding protein 1 (PABPC1) expression.

Total RNA was extracted and genomic DNA was eliminated with a Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare Life Sciences, Buckinghamshire, UK). One microgram of DNase I-treated total RNA was reverse-transcribed with a RevertAid H Minus First Strand cDNA Synthesis Kit (Life Technologies Corporation, Carlsbad, CA, USA) into cDNA. It was then used as the template for real-time PCR reactions and analysis. The real-time PCR reactions were conducted using a QuantiNovaTM SYBR® Green PCR Kit (QIAGEN, Valencia, CA, USA) on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). This involved an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturing at 95 °C for 5 s, and combined annealing/extension at 60 °C for 10 s, as described in the manufacturer’s instructions. The primer sequences were as follows: 18S rRNA forward 5′-GTAACCCGTTGAACCCCATT-3′ and reverse 5′-CCATCCAATCGGTAGTAGCG-3′; TP53 forward 5′-AAGTCTAGAGCCACCGTCCA-3′ and reverse 5′-CAGTCTGGCTGCCAATCCA-3′; TGF-β forward 5′-TACAGACCCTACTTCAG-3′ and reverse 5′-AAATCTTGCTTCTAGTT-3′. The relative gene expression, normalized to the internal control 18S rRNA, was calculated based on the ΔΔCT method, and the fidelity of the PCR reactions was determined by melting temperature analysis.

Total RNA was isolated from 150 EOC tissue samples using TRIZOL reagent (Generay Biotech, Co., Ltd., Shanghai, China) according to the manufacturer’s protocols. The cDNA was then synthesized using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The PCRs were carried out using a QuantiNova TMSYBR® Green PCR Kit (Qiagen, Hilden, Germany). Relative expression levels of hMSH2 were calculated with the 2^–ΔCt method using GAPDH as an endogenous control, and all experiments were repeated three times.