Chamq sybr qpcr master mix

Manufactured by Vazyme

Sourced in China, United States, Switzerland, Germany, Japan, Canada, Australia

ChamQ SYBR qPCR Master Mix is a ready-to-use reagent for real-time quantitative PCR (qPCR) amplification and detection of DNA targets using SYBR Green I as the fluorescent dye. It contains all the necessary components, including a high-performance DNA polymerase, SYBR Green I, and optimized buffer system, to enable efficient and sensitive qPCR reactions.

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906 protocols using chamq sybr qpcr master mix

Quantifying Melanoma Stem Cell mRNA

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Melanoma stem cells and non-stem cells were sorted in our laboratory previously from cell line MDA-MB-435 [24] or A375 [25] . Melanoma stem cells and non-stem cells were cultured in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (EGF) (Beyotime Biotechnology, China), 10 ng/mL basic broblast growth factor (bFGF) (Beyotime Biotechnology), 5 mg/mL of insulin (Beyotime Biotechnology), and 2% of B-27 (Sigma, USA) at 37℃ in a humidi ed atmosphere with 5% CO 2 .
Quanti cation of mRNA with real-time PCR Total RNAs were extracted from cells using an RNA Isolation Kit (Ambion, USA). The reverse transcription reaction was conducted with PrimeScript RT Reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using 2×ChamQ SYBR qPCR Master Mix (Vazyme, USA). The PCR reaction mixture (10 μL) contained Rox reference Dye, cDNA, ChamQ SYBR qPCR Master Mix (Vazyme) and primers (Table S1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was included for normalization. The 2 -(△△Ct) method was used to calculate the relative fold change of mRNA expression [26] . PCR was conducted by maintaining the reaction at 95℃ for 30 s, and then alternating for 40 cycles between 95℃ for 5 s and 60℃ for 30 s.

Chu M., Wan H., & Zhang X. (2020). Requirement of splicing factor hnRNP A2B1 of hnRNPs for tumorigenesis of melanoma stem cells.

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Comprehensive RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted from cells with TRIzol (Invitrogen) and reverse-transcribed with oligo dT (Takara) and RT-ACE (Toyobo) to generate complementary DNA (cDNA). Then, quantitative real-time PCR (qRT-PCR) was performed with ChamQ SYBR qPCR Master Mix (Vazyme) and CFX96 qRT-PCR machine (Bio-Rad). Human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used to normalize qRT-PCR of these samples.
For miRNA, total RNA of cells was reverse-transcribed using specific stem-loop primers (31 (link)). The qRT-PCR for miRNA detection was carried out with ChamQ SYBR qPCR Master Mix (Vazyme) and a CFX96 qRT-PCR machine (Bio-Rad). U6 small nucleolar RNA was used for normalization.
All reactions were analyzed with three repeats. All primer sequences were listed in table S1.

Wei Y., Wang T., Ma L., Zhang Y., Zhao Y., Lye K., Xiao L., Chen C., Wang Z., Ma Y., Zhou X., Sun F., Li W., Dunk C., Li S., Nagy A., Yu Y., Pan G., Lye S.J, & Shan Y. (2021). Efficient derivation of human trophoblast stem cells from primed pluripotent stem cells. Science Advances, 7(33), eabf4416.

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RT-qPCR Gene Expression Analysis

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The 20-µl total reaction volume were configured according to the protocol of ChamQ SYBR qPCR Master Mix (Vazyme), contained 10-µl 2 × ChamQ SYBR qPCR Master Mix, 0.4 µl (10 μM) of each gene specific primer, 1 µl of cDNA, and 8.2 µl of ddH₂O. The amplification reaction program was set as follows: pre-denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s. The melting curves were analyzed in the 60–95°C temperature range after amplification step. The reaction was performed on a Roche LightCycler96 instrument to obtain CT values, amplification curves, melting curves, and standard curves. All samples were carried out in four technical and three biological replicates, and the negative control (no template) was performed in parallel.

Wang X., Kong X., Liu S., Huang H., Chen Z, & Xu Y. (2020). Selection of Reference Genes for Quantitative Real-Time PCR in Chrysoperla nipponensis (Neuroptera: Chrysopidae) Under Tissues in Reproduction and Diapause. Journal of Insect Science, 20(4), 20.

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Quantification of Viral RNA and DNA in SIV Studies

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The viral load, represented by the SIV RNA copies in plasma, was detected as we previously described (53 (link)). RNA was extracted by a QIAamp viral RNA mini kit (Qiagen, Cat. No. 52906) and was quantified using the QuantiTect SYBR Green RT‒PCR Kit (Qiagen, Cat. No. 204245). For cell-associated RNA detection, RNA from PBMCs was separated using the EastepTM Super Total RNA Extraction Kit (Promega, Cat. No. LS1040) and then reverse transcribed using GoScript Reverse Transcription Mix, Random Primers (Promega, Cat. No. A2800). The first round of PCR was performed using Premix Taq (Takara, Cat. No. R004A). The following second real-time fluorescent quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Cat. No. Q311-02). For SIV total gag DNA detection, genomic DNA was extracted from PBMCs by a Rapid Genomic DNA Kit (Biomed, Cat. No. DL110-01) and was quantified using ChamQ SYBR qPCR Master Mix (Vazyme, Cat. No. Q311-02). All final data were collected and analyzed using a CFX96 Real-Time PCR system (Bio-Rad).

He Y., Wu C., Liu Z., Zhang Y., Feng F., Lin Z., Wang C., Yang Q., Wen Z., Liu Y., Zhang F., Lin Y., Zhang H., Qu L., Li L., Cai W., Sun C., Chen L, & Li P. (2023). Arsenic trioxide-induced apoptosis contributes to suppression of viral reservoir in SIV-infected rhesus macaques. Microbiology Spectrum, 11(5), e00525-23.

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Quantification of Viral RNA and DNA in SIV Studies

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Quantifying mRNA Expression with Real-Time PCR

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Total RNAs were extracted from cells using an RNA Isolation Kit (Ambion, USA). The reverse transcription reaction was conducted with PrimeScript RT Reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using 2 × ChamQ SYBR qPCR Master Mix (Vazyme, USA). The PCR reaction mixture (10 μL) contained Rox reference Dye, cDNA, ChamQ SYBR qPCR Master Mix (Vazyme), and primers (Table S1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene for normalization. The 2^-(△△Ct) method was used to calculate the relative fold change of mRNA expression [20 (link)]. PCR was conducted by maintaining the reaction at 95 °C for 30 s and then alternating for 40 cycles between 95 °C for 5 s and 60 °C for 30 s.

Chu M., Wan H, & Zhang X. (2021). Requirement of splicing factor hnRNP A2B1 for tumorigenesis of melanoma stem cells. Stem Cell Research & Therapy, 12, 90.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA of tissue (each 100 mg) and cells were extracted using Invitrogen Trizol Reagent (Invitrogen) following the manufacturer. The RNA quality was assessed by denaturing formaldehyde agarose gel, in which the 28S:18S ratio of 1.8–2.1 were considered to be qualified. Total RNA (0.5 μg) was reverse transcribed to cDNA in a total volume of 8 μL using HiScript^®II Q RT SuperMix for qPCR (Vazyme, Nanjing, China) and then the cDNA was diluted by three times. qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme). The sequences of primers are shown in Table 1. qRT-PCR was performed using the Eppendorf Mastercycler^® ep realplex (Eppendorf, Hamburg, Germany), in a total volume of 20 μL, containing 2 μL of cDNA sample, 0.2 μM of each primer and 1 × ChamQ SYBR qPCR Master Mix (Vazyme). The PCR conditions were 95°C for 10 min and 35 cycles of 95°C for 15 s, 60°C for 30 s. Relative mRNA levels were normalized to endogenous reference gene (NONO or TBP), and was calculated using the formula 2^–ΔΔCt (Livak and Schmittgen, 2001 (link)). Each assay was performed in three independent experiments with three replicates in each.

Sun Y., Jin Z., Zhang X., Cui T., Zhang W., Shao S., Li H, & Wang N. (2020). GATA Binding Protein 3 Is a Direct Target of Kruppel-Like Transcription Factor 7 and Inhibits Chicken Adipogenesis. Frontiers in Physiology, 11, 610.

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Quantitative RT-PCR Analysis of Gene Expression

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RNA simple Total RNA Kit (Tiangen, China) was used to extract total RNA from samples. HiScript^® III Q RT Super-Mix for qPCR Kit (Vazyme, China) reverse transcribed 1μg total RNA into cDNA for gene cloning and qPCR. Based on the selected gene sequence, the primer pair (Supplemental Table 1) was designed using Beacon Designer™ 8.10 (Premier Biosoft International, USA). qRT-PCR was conducted on Quant-Studio™ 5 Real-Time PCR System (Applied Biosystems) with ChamQ SYBR qPCR Master Mix (Vazyme, China), which included 10 μL of ChamQ SYBR qPCR Master Mix (2 ×), 0.4 μL of sense or anti-sense primer, 0.4 μL of ROX Reference dye (50 ×), 1 μL of cDNA and 7.8 μL of ddH₂O in a total volume of 20 μL. The PCR program was as follows: 95°C for 30 s; 40 cycles of 95°C for 10 s, 58°C for 10 s; and finally, 72°C for 30 s.
The relative expression levels of the selected genes were calculated by 2^-ΔΔCT method using cucumber or tobacco actin genes as internal controls (Livak and Schmittgen, 2001 (link)).

Zhu M., Chen G., Wu J., Wang J., Wang Y., Guo S, & Shu S. (2023). Identification of cucumber S-adenosylmethionine decarboxylase genes and functional analysis of CsSAMDC3 in salt tolerance. Frontiers in Plant Science, 14, 1076153.

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Quantitative RNA Expression Analysis

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Total RNA was extracted from leaves using TransZol reagent (TRANSGEN, China, Beijing) following the manufacturer’s instructions. Total RNA was treated with DNase I (Thermo, Waltham, USA) to remove the contaminated genomic DNA. The cDNA was synthesized using TransScript cDNA Synthesis SuperMix (TRANSGEN, Beijing, China). All reactions were performed using ChamQ SYBR qPCR Master Mix (Vazyme biotech, Nanjing, China) with a reaction system containing 10.0 μL of 2 × ChamQ SYBR qPCR Master Mix, 0.4 μL of primers, 1.0 μL of cDNA, and 8.2 μL of ddH₂O. Quantitative real-time PCR (qRT-PCR) was conducted on QuantStudioTM 6 Real-Time PCR System (Thermo). The reactions were performed with three biological replicates and three technical replicates. The transcript levels of genes in different individuals were analyzed using the 2^−ΔΔCT method and normalized with actin as an internal control. (Livak and Schmittgen 2001 ). The primers used for qRT-PCR assays were listed in the supplemental dataset 3.

Tang Q., Kuang H., Yu C., An G., Tao R., Zhang W, & Jia Y. (2021). Non-vernalization requirement in Chinese kale caused by loss of BoFLC and low expressions of its paralogs. TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik, 135(2), 473-483.

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Quantifying Viral DNA Load by qPCR

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The viral tissue load was measured by absolute quantitative PCR (qPCR) targeting the gB gene. The viral DNA from tissues were extracted by TIANamp virus DNA/RNA kit (Tiangen, Beijing, China) with the ChamQ^TM SYBR^® qPCR Master Mix (Vazyme, Nanjing, China) on an ABI 7500 Real-time PCR system (Applied Biosystem, United States) according to the manufacturer’s recommendations. The primers were as follows: upstream primer: 5′-GTCTGTGAAGCGGTTCGTGAT-3′ and downstream primer: 5′-ACAAGTTCAAGGCGCACATCTAC-3′. Seven serial dilutions of plasmid containing gB with the copy number from 10¹ to 10⁷ copies/μL served as template to generate a standard curve. The PCR was performed in a 20 μL reaction containing 0.4 μL gene specific primers (10 μM), 10 μL ChamQ^TM SYBR^® qPCR Master Mix (Vazyme, Nanjing China), 2 μL PRV genome, and 7.2 μL ddH₂O. The PCR parameter was set up as follows: 50°C for 2 min, 95°C for 2 min; 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. The viral loads were calculated with the 7500 System SDS software according to standard curve and expressed as log₁₀ copies per gram of tissue sample.

Ren J., Wang H., Zhou L., Ge X., Guo X., Han J, & Yang H. (2020). Glycoproteins C and D of PRV Strain HB1201 Contribute Individually to the Escape From Bartha-K61 Vaccine-Induced Immunity. Frontiers in Microbiology, 11, 323.

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