The gene expression of enzymes involved in the glutathione synthesis and apoptosis markers were assessed in liver samples. The RNA extraction was performed using Trizol reagent following the manufacturer's instructions (Ludwig-Biotec, São Paulo, Brazil). The RNA extracted was measured by a Thermo Scientific NanoDropTM 1000 spectrophotometer. To perform reverse transcription, RNA was added to the samples of RNA (1000 ng/μl) with 0.2 μl of DNAase (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C for 5 minutes followed by heating at 65°C for 10 minutes. The cDNA was generated with Iscript cDNA and Mix Iscript (Bio-Rad Laboratories, Hercules, CA, USA). qRT-PCR was conducted in the Rotor-Gene Q 5plex HRM System (Qiagen Biotechnology, Germany) with 2x QuantiFast SYBR Green PCR Master Mix (Qiagen Biotechnology, Germany). qRT-PCR reactions were run in triplicate, using 1 µM of each primer, 1000 ng/µL of cDNA, RNAase-free water, and 2x QuantiFast SYBR® Green PCR Master Mix (Qiagen Biotechnology, Germany). β-Actin was used as the housekeeping gene, and its expression level was used as an internal control. The relative gene expression was calculated using the comparative cytosine-thymine (Ct) method and was expressed as the fold expression compared to the control. The specific primer pairs used are described in Table 2 .
Quantifast sybr green pcr master mix
Manufactured by Qiagen
Sourced in Germany, United States, Switzerland, Sweden, United Kingdom
The QuantiFast SYBR Green PCR Master Mix is a ready-to-use solution for performing real-time PCR with SYBR Green detection. It contains all the necessary components, including a DNA polymerase, SYBR Green I dye, and optimized buffer system, to enable efficient and sensitive amplification and detection of DNA targets.
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Lab products found in correlation
Quantitect reverse transcription kit, by Qiagen (16 mentions)
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Lightcycler 480, by Roche (10 mentions)
Rneasy mini kit, by Qiagen (10 mentions)
Lightcycler 480 2 real time pcr instrument, by Roche (9 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Mirneasy mini kit, by Qiagen (6 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (5 mentions)
Icycler iq detection system, by Bio-Rad (5 mentions)
Quantitect primer assay, by Qiagen (5 mentions)
Rna extraction kit, by Qiagen (5 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (4 mentions)
Sybr green, by Bio-Rad (4 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
119 protocols using quantifast sybr green pcr master mix
1
Glutathione Synthesis and Apoptosis in Liver
2
Quantification of tRNA-derived small RNA levels
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In order to measure the tsRNA level after transfection, the 15–40 nt small RNA fraction was eluted from a denaturing polyacrylamide gel (see Gebert et al. 2015 (link)) and polyadenylated using the A-Plus Poly(A) Polymerase Tailing Kit (Cellscript). After ethanol precipitation the poly(A) tailed RNA was reversely transcribed with the SuperScript IV reverse transcriptase (Thermo Fisher) using the RT-primer 5′-CGAATTCTAGAGCTCGAGGCAGGCGACATGT25VN-3′. For qPCR 1 µL of cDNA was mixed with 0.5 µL 10 µM sequence-specific forward primer, 0.5 µL 10 µM RT-primer-specific reverse primer, 3 µL water and 5 µL 2× QuantiFast SYBR Green PCR Master Mix (Qiagen). Technical duplicates of this reaction mix were then analyzed on a Corbett Rotor-Gene 6000 real-time PCR cycler. Finally, the copy numbers of the respective tsRNAs were quantified by standard curves of the individual primer pair amplicons. As normalizers the miRNAs miR25, miR532 and miR99a were used. The boxplots were created with R using the R packages ggplot2 and Cairo (version 3.4.3). qPCR primer sequences are available in Supplemental Table S3 .
3
RNA Extraction and Real-time PCR Analysis
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Total RNA was extracted from cells by RNA kit II (Omega Bio-Tec Inc., USA) following manufacturer’s instructions and quantified with Nano Drop spectrophotometry (BioTek, USA). We used 2 µg total RNA to synthesize first strand cDNA by RevertAid first strand cDNA synthesis kit (Thermo Scientific). Real-time PCR was performed on Bio-Rad CFX Connect™ Real-time System (Bio-Rad, USA) and amplified with an optimized cycling condition: 5 min at 95 °C, then 10 s at 95 °C and 30 s at 60 °C for 38 cycles. The total reaction system was 20 µL: 10 µL 2× QuantiFast SYBR Green PCR MasterMix (Qiagen, Germany), 1 µL of cDNA templates, 0.5 µM primer and 8 µL of DNAase-RNase Free water. The primers were provided by Shenggong Company (Shanghai, China) and the sequences were used in our previous studies (8 (link),17 (link)).
4
Quantitative Real-Time PCR Reaction Protocol
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Real-time PCR reactions were performed in 96-well plates with a Bio-Rad CFX96TM Real-time PCR Detection System. Reactions were carried out in a final volume of 25 μl containing 12.5μl 2× QuantiFast SYBR Green PCR Master Mix (Qiagen), 500nM of each primer, 1μl cDNA and 9.5μl DNase/RNase-free water. The cycling program was 95°C for 5 min to activate the polymerase followed by 40 cycles of denaturation at 95°C for 10s, annealing at 60°C for 20s and extension at 72°C for 20s. Melting curve analysis was conducted by heating samples from 65°C to 95°C with continuous fluorescent acquisition. All reactions were performed in triplicate for each cDNA sample. In addition, no template controls were tested for each primer pair. Standard curves were established using a 10-fold dilution series of purified PCR fragments as templates.
5
Real-Time qPCR Analysis of Plant Gene Expression
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Plant tissues were immediately frozen in liquid nitrogen and stored at −80 °C. RNA isolation, DNAse I treatment and first-strand cDNA synthesis were performed as described [60 (link)]. Real-time PCR was carried out in a final volume of 12.5 microl, containing the 6.5 μL of 2× QuantiFast SYBR Green PCR Master Mix (Qiagen), the primer pairs (300 nM) and the cDNA template. Amplifications were performed as reported [61 (link)]. Primers and their temperature of annealing are presented in Supplementary Table S1 . Real-time RT-PCR was performed in three biological replicates. Reactions were carried out in three technical replicates in an ABI 7900 HT thermocycler (Applied Biosystems, Foster City, CA, USA). For quantification of gene expression, we employed the DeltaDeltaCt method [62 (link)], using the GAPDH as reference gene and the plants treated with the 20:80 ratio as calibrator genotype. Fold change are expressed in relative quantities (RQ) compared to the calibrator condition, set as 1. Significant differences were assessed with an analysis of variance, followed by Duncan’s post-hoc test for multiple comparisons of the 2−ΔCt values.
6
Quantitative PCR for mRNA Expression
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Real-time quantitative PCR method was applied to validate the expression level of mRNAs. Briefly, approximately 0.5 μg of the same RNA samples was used for reverse transcription using GeneAmp® PCR System 9700 (Applied Biosystems, USA) to synthesize cDNA templates. qRT-PCR analysis was performed in triplicate with a LightCycler® 480 II Real-time PCR Instrument (Roche, Swiss) using QuantiFast® SYBR® Green PCR Kit (Qiagen, Germany) according to the manufacturer’s instructions. The reaction system was consisting of 1 μL cDNA (1:4 dilution), 5 μL of 2× QuantiFast® SYBR® Green PCR Master Mix (Qiagen, Germany), 0.2 μL of forward primer, 0.2 μL of reverse primer, and 3.6 μL of nuclease-free water. The reaction condition included initial denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and a final stage of dissociation analysis. The primer sequences were designed in the laboratory and synthesized by Generay Biotech (shanghai, China) based on the mRNA sequences obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/ ). Bovine β-actin gene was used as an internal reference and the 2−ΔΔCt algorithm was applied to calculate the relative expression level of genes between samples.
7
Quantitative Real-Time PCR Analysis of Gene Expression
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Real-time PCR of target gene copy numbers in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts was carried out using individual primers mentioned in Table 1 . Gene expression levels were quantified using a SYBR Green kit (Qiagen, Germany). Data were analyzed using MxPro quantitative PCR Software (Agilent technologies). Table 1 lists the oligonucleotide primer sequences used for GAPDH, VEGF, caspase-3, caspase-9, Bax, Bcl2, and IL-6 (Hua et al. 2015 (link); Li et al. 2014 (link)).
Primer’s sequence of GAPDH, caspase-3, caspase-8, Bax, and Bcl2 genes
| Target | Sequence |
|---|---|
| GAPDH | F: 5′- TGACATCAAGAAGGTGGTGA-3′ R: 5′- TCATACCAGGAAATGAGCTT-3′ |
| Caspase-3 | F: 5′-AATTCAAGGGACGGGTCATG-3′ R: 5′-TGACACAATACACGGGATCTG -3′ |
| Caspase-8 | F: 5′-GATGAGGCAGACTTTCTGCT -3′ R: 5′-CATAGTTCACGCCAGTCAGGAT -3′ |
| Bax | F: 5′-GGCGAATTGGAGATGAACTG -3′ R: 5′-TGCCATCAGCAAACATGTCA -3′ |
| Bcl2 | F: 5′-ATCCAGGATAACGGAGGCTG-3′ R: 5′-CAGGTATGCACCCAGAGTGA -3′ |
8
Quantitative RNA Expression Analysis
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Total RNA was extracted from colon tissue using TRIzol (Invitrogen, Carlsbad, CA, USA). mRNA was reverse transcribed using SYBR Premix Ex Taq™ II (Takara, Dalian, China). Real-time PCR was performed using an Applied Biosystems HT7900 PCR system with 2× QuantiFast SYBR Green PCR Master Mix (QIAGEN, Hilden, Germany), primers (1 μM; Table S1 ) and <100 ng cDNA in a 25 μl reaction mixture. The relative expression of target genes was analysed using the ∆∆Ct method.
9
Gene Expression Analysis of MG63 Cells
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24 h after CAP application total RNA of MG63 cells was extracted using an RNA extraction kit (Qiagen, Hilden, Germany). iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, Munich, Germany) was used to reversely transcribe 1 μg of RNA at 42 °C for 90 min followed by 85 °C for 5 min. 1 μl of cDNA was amplified as a template in a 25 μl reaction mixture containing 12.5 μl 2 × QuantiFast SYBR Green PCR Master Mix (Qiagen), 2.5 μl of primers (0.5 μM each), and 9 μl deionized water. The mixture was heated at 95 °C for 5 min initially, followed by 40 cycles with denaturation at 95 °C for 10 s and combined annealing/extension at 60 °C for 30 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. mRNA expression of IL-1β, IL-6, IL-8, TNFα, COX2, COL1α, MMP1, Ki67, PCNA and CCL2 was detected by real-time PCR using the iCycler iQ™ detection system (Bio-Rad Laboratories, Hercules, CA, USA), SYBR Green (Bio-Rad Laboratories), and specific primers (QuantiTect Primer Assay, Qiagen). mRNA was quantified by the comparative threshold cycle method.
10
Quantifying Gene Expression in Grape Plants
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Total RNA isolated from leaves of the untransformed and MrRPV1-transgenic Shiraz plants, mock-inoculated or inoculated with P. viticola, was used for cDNA synthesis using HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co. Ltd. Nanjing, China). Real-time PCR was performed using a LightCycler® 480 II Real-time PCR Instrument (Roche, Switzerland) with a 10 μl PCR mixture that included 1 μl of cDNA, 5 μl of 2× QuantiFast® SYBR® Green PCR Master Mix (Qiagen, Germany), 0.2 μl of forward primer, 0.2 μl of reverse primer and 3.6 μl of nuclease-free water. Reactions were incubated in a 384-well optical plate (Roche, Switzerland) at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Each sample was run in triplicate. Specific primers for the selected genes are listed in Supplemental Table 1 . The expression levels of mRNAs were normalized to that of EF1α and were calculated using the 2−ΔΔCt method75 (link).
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