Sybr pcr master mix

Manufactured by Roche

Sourced in United States, Sweden, Switzerland

SYBR PCR Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis. It contains all the necessary components, including a DNA polymerase, SYBR Green I dye, and buffer, to perform qPCR reactions.

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SYBR Green Real Time PCR master mixes SYBR Master Mix PCR Master Mix

11 protocols using sybr pcr master mix

Analysis of ER Stress Markers in HGECs

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HGECs were suspended in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and stored at - 80°C.
Total RNA was puri ed using TRIzol reagent following the manufacturer's instructions and quanti ed with a Nanodrop 2000. cDNA was synthesized from 500 ng of total RNA using Prime Script RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). Real-time PCR was performed in a 96-well optical reaction plate using SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). mRNA expression was assayed on a Bio-Rad CFX96 detection system (Roche, Sweden). Details of the RT-qPCR primers used in this experiment are shown in Table 1. Relative SERPINH1, GRP78, IRE1, TRAF2, NLRP3, IL-1β, XBP1-s and XBP1-u expression was quanti ed with the 2-ΔΔCt method using GAPDH expression as an endogenous control.

Li M., Huang S., Zhang Y., Song Z., Fu H., Lin Z, & Huang X. (2022). Regulation of the unfolded protein response transducer IRE1α by SERPINH1 aggravates periodontitis with diabetes mellitus via prolonged ER stress. Cellular signalling, 91.

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T cell gene expression analysis

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T cells labeled with CellTracker dyes before coculture were harvested and then rinsed gently using the tip. Next, labeled T cells were sorted directly into TRIzol for qPCR. Total RNA was extracted using TRIzol reagent (Molecular Research Center, Inc.) according to the manufacturer's instructions. Reverse transcription was performed using oligo-dT primers (Thermo), and quantitative real-time qRT-PCR was performed using SYBR PCR Master Mix (Roche) according to the manufacturer's instructions. qRT-PCR was conducted in triplicate for each sample, and three independent experiments were performed. Signals were detected using a Light Cycler 480 detection system (Roche). The primer sequences are listed in Supplementary Table 1.

Zheng S., Huang K., Xia W., Shi J., Liu Q., Zhang X., Li G., Chen J., Wang T., Chen X, & Xiang A.P. (2021). Mesenchymal Stromal Cells Rapidly Suppress TCR Signaling-Mediated Cytokine Transcription in Activated T Cells Through the ICAM-1/CD43 Interaction. Frontiers in Immunology, 12, 609544.

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Quantitative RT-PCR Assay for miRNA and mRNA

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The total RNA of cells and tissues was extracted with a TRIzol Kit (Invitrogen), and the first strand cDNA was reversely transcribed according to the instructions of a cDNA Reverse Transcription Kit (Takara, China). The primers were synthesized and provided by RiboBio (Guangzhou, Chian). SYBR PCR Master Mix (Roche) was used for qRT-PCR. The PCR program setup referred to our previous study (Li et al., 2020 (link)). The internal reference of miR-327 is U6 and that of FGF10 is GAPDH. The primer sequences were as follows:

Rno-miR-327 loop-primer 5′-GTCGTATCCAGTGCAG GGTCCGAGGTATTCGCACTGGATACGACACCCTCAT-3′

Forward primer 5′-TGCGCCCTTGAGGGGCATG-3′, Reverse primer 5′-CCAGTGCAGGGTCCGAGGTATT-3’;

U6: Forward primer 5′-CGCTTCGGCAGCACATATAC-3′, Reverse primer 5′-AAATATGGAACGCTTCACGA-3’;

FGF10: Forward primer 5′-CAACGGCAGGCAAATGTATG-3′, Reverse primer 5′-AGGAAGTGAGCGGAGGTGTT-3’;

GAPDH: Forward primer 5′-GAACGGGAAGCTCACTGG-3′,

Reverse primer 5′-GCCTGCTTCACCACCTTCT-3’.

Zheng T., Yang J., Zhang J., Yang C., Fan Z., Li Q., Zhai Y., Liu H, & Yang J. (2021). Downregulated MicroRNA-327 Attenuates Oxidative Stress–Mediated Myocardial Ischemia Reperfusion Injury Through Regulating the FGF10/Akt/Nrf2 Signaling Pathway. Frontiers in Pharmacology, 12, 669146.

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Quantifying Gene and miRNA Expression

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Total RNA was extracted from periodontal tissue and cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was generated with PrimeScript RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). The expression level of genes was measured by qPCR in a Bio-Rad CFX96™ Detection System (Roche, Sweden) with SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). Small RNA was extracted from cells with an miRNA isolation kit (Qiagen, Hilden, Germany), and cDNA was generated with an miRNA reverse transcription kit (Shenggong, Shanghai, China). The expression level of miRNAs was measured by qPCR in a Bio-Rad CFX96™ Detection System with SYBR PCR Master Mix. U6 was used as the internal reference. The primers used are shown in Supplementary Table S1.

Shen Z., Kuang S., Zhang Y., Yang M., Qin W., Shi X, & Lin Z. (2020). Chitosan hydrogel incorporated with dental pulp stem cell-derived exosomes alleviates periodontitis in mice via a macrophage-dependent mechanism. Bioactive Materials, 5(4), 1113-1126.

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RNA Extraction and qRT-PCR Analysis in 3T3-L1 Adipocytes

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Total RNA was extracted from 3T3-L1 adipocytes with total RNA genes extraction kit according to manufacturer’s protocol. The cDNA synthesis from the isolated RNA was performed using a reverse transcriptional system. The primer sets are shown in Table 1. Quantitative real-time PCR was performed in a reaction containing cDNA and SYBR PCR master mix (Roche Group, Switzerland). Samples were analyzed with the ABI PRISM 7500 sequence detection system (Applied BioSystems,USA). All PCRs were performed in triplicate, and the specificity of the reaction was determined by melting curve analysis at the dissociation stage.

He Q., Yang Q.C., Zhou Q., Zhu H., Niu W.Y., Feng J., Wang Y., Cao J, & Chen B.Y. (2014). Effects of Varying Degrees of Intermittent Hypoxia on Proinflammatory Cytokines and Adipokines in Rats and 3T3-L1 Adipocytes. PLoS ONE, 9(1), e86326.

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RNA Isolation and RT-qPCR Analysis of Periodontal Tissue

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Total RNA was purified from periodontal tissue with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was generated using PrimeScript RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). The cells isolated by FACS were lysed, and reverse transcription was performed using a SYBR™ Green Fast Advanced Cells-to-CT™ Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Gene expression levels were measured by RT-qPCR using a BioRad CFX96^TM Detection System (Roche, Sweden) and SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). The primers used in this experiment are shown in Supplementary Table 2.

Shen Z., Kuang S., Zhang M., Huang X., Chen J., Guan M., Qin W., Xu H.H, & Lin Z. (2020). Inhibition of CCL2 by bindarit alleviates diabetes-associated periodontitis by suppressing inflammatory monocyte infiltration and altering macrophage properties. Cellular and Molecular Immunology, 18(9), 2224-2235.

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Quantifying Gene Expression in Testis

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Total RNA from the testes or cells was extracted using a RNeasy mini kit (Qiagen, Dusseldorf, Germany) according to the manufacturer's protocol. Reverse transcription was performed using a High‐Capacity RNA‐to‐cDNA^TM Kit (Thermo Fisher). Quantitative RT‐PCR was performed using SYBR PCR Master Mix (Roche, Basel, Switzerland) and a Light Cycler 480 Detection System (Roche). To check primers, a melting curve was generated to confirm a single peak and rule out the possibility of non‐specific product or primer‐dimer formation. β‐actin was amplified as internal control, and the target gene expression was presented as ratio to WT mice or WT‐SLCs. The primers used for quantitative RT‐PCR were listed at Table S2 (Supporting Information).

Xia K., Wang F., Tan Z., Zhang S., Lai X., Ou W., Yang C., Chen H., Peng H., Luo P., Hu A., Tu X., Wang T., Ke Q., Deng C, & Xiang A.P. (2023). Precise Correction of Lhcgr Mutation in Stem Leydig Cells by Prime Editing Rescues Hereditary Primary Hypogonadism in Mice. Advanced Science, 10(29), 2300993.

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Quantifying Target Gene Expression

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Total RNA isolation and cDNA synthesis by reverse transcription were performed using the TRIzol reagent and a reverse transcriptase kit (Thermo Fisher Scientific, Boston, MA, USA). The mRNA levels of individual genes were determined using a SYBR PCR Master Mix (Roche, Basel, Switzerland) in the ABI StepOne Plus™ Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Data was analyzed by the 2−ΔΔCt method and normalized based on GAPDH as the reference gene. The primers used in this experiment are listed in Table 1.

Liu H., Kai L., Du H., Wang X, & Wang Y. (2019). LPS Inhibits Fatty Acid Absorption in Enterocytes through TNF-α Secreted by Macrophages. Cells, 8(12), 1626.

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Total RNA Extraction and RT-qPCR Protocol

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Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed using ReverTra Ace (Toyobo Co, Ltd, Osaka, Japan), and RT-qPCR was performed using SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). Three independent experiments were performed. The signals were detected using a Light Cycler 480 Detection System (Roche, Sweden). The primer sequences are listed in Supplementary Table 1.

Shen Z., Wang J., Huang Q., Shi Y., Wei Z., Zhang X., Qiu Y., Zhang M., Wang Y., Qin W., Huang S., Huang Y., Liu X., Xia K., Zhang X, & Lin Z. (2018). Genetic modification to induce CXCR2 overexpression in mesenchymal stem cells enhances treatment benefits in radiation-induced oral mucositis. Cell Death & Disease, 9(2), 229.

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RNA Extraction and Quantitative RT-PCR Analysis

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Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. RNA purity was determined by 260/280 ratio (1.87–1.93) and 260/230 ratio (2.18–2.34) using NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA). Reverse transcription was performed using a High-Capacity RNA-to-cDNA^TM Kit (Thermo Fisher Scientific). Quantitative RT–PCR was performed using SYBR PCR Master Mix (Roche) and a Light Cycler 480 Detection System (Roche). To check primers, a melting curve was generated to confirm a single peak and rule out the possibility of non-specific product or primer–dimer formation. GAPDH was amplified as control, and the target gene expression levels were calculated using the ΔCt methods and expressed as relative expression. The primers are listed in Supplementary Table SI.

Xia K., Ma Y., Feng X., Deng R., Ke Q., Xiang A.P, & Deng C. (2020). Endosialin defines human stem Leydig cells with regenerative potential. Human Reproduction (Oxford, England), 35(10), 2197-2212.

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