Sybr green real time master mix

Manufactured by Thermo Fisher Scientific

Sourced in United States, Lithuania, United Kingdom

SYBR Green Real-Time Master Mix is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I dye, DNA polymerase, and necessary reaction components.

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17 protocols using sybr green real time master mix

Hippocampal Gene Expression Analysis

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Total RNA was isolated using the Trizol reagent (Invitrogen) from micro-dissected hippocampus and 5 µg was used to synthesize cDNA using M-MLV reverse transcriptase following the manufacturer’s protocol (Invitrogen). RT-PCR reactions were performed using SYBR^® Green Realtime Master Mix and ABI PRISM^® 7700 (Applied Biosystems). The primer sequences for the genes analyzed are summarized in Table S2. Each sample was run in duplicate and repeated twice. Threshold cycle values were used to calculate the fold change in the transcript levels by using the 2^−ΔΔCt method. The relative mRNA expression levels were normalized to the tubulin gene.

Jin H.J., Pei L., Li Y.N., Zheng H., Yang S., Wan Y., Mao L., Xia Y.P., He Q.W., Li M., Yue Z.Y, & Hu B. (2017). Alleviative effects of fluoxetine on depressive-like behaviors by epigenetic regulation of BDNF gene transcription in mouse model of post-stroke depression. Scientific Reports, 7, 14926.

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Soybean Lipoxygenase Gene Expression

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The expression level of Lipoxygenase 1 (LOX1) and Lipoxygenase 2 (LOX2) genes in soybean seeds was determined. Briefly, total RNA was isolated using the RNeasy Plant Mini kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer’s protocol. RNA concentration was determined with a fluorometer Qubit™ (Invitrogen, Carlsbad, CA, USA), and quality and quantity were assessed spectrophotometrically before the cDNA was synthesized using the Thermo Scientific RevertAid Reverse Transcriptase in a Bio-Rad My Cycler™ Termal Cycler.
An ABI 7500 Fast Real-Time PCR system was used to perform the qRT-PCR (Applied Biosystems, Foster City, CA, USA) with the SYBR Green Real-time Master Mix. The LOX1 and LOX2 primer sequences in this study are detailed in Table 1. The housekeeping soybean elongation factor (ELF1b) was used as a reference gene for relative quantification and the target expression relative to the housekeeping was used for ANOVA.

Sabljic I., Barneto J.A., Balestrasse K.B., Zavala J.A, & Pagano E.A. (2020). Role of reactive oxygen species and isoflavonoids in soybean resistance to the attack of the southern green stink bug. PeerJ, 8, e9956.

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Quantitative Analysis of MUC5AC mRNA

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Well-differentiated HBECs and lung tissues were harvested for RNA extraction using TRIzol reagent (Invitrogen, USA). Total RNA was DNase treated (Invitrogen, USA) before cDNA synthesis. Reverse transcription of RNA was conducted with EvoScript Universal cDNA Master kit (Roche, Basel, Switzerland) following the manufacturer's instruction. Quantitative RT-PCR assay was performed using SYBR Green Real-Time Master Mix (Applied Biosystems, Lithuania) based on the manufacturer's protocol. Relative quantity of mRNA was obtained by using the comparative Ct method and normalized by housekeeping genes such as human ribosomal protein S13 (RPS13) or rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer for human MUC5AC is as follows: 5′-CTT TGG CAT CTG TGA GGA GC-3′ (forward primer) and 5′-CAC AGA AGC AGA GGT CTT GC-3′ (reverse primer). The primer for rat MUC5AC is as follows: 5′-AAC TCT GCC CAC CAC AAG C-3′ (forward primer) and 5′-TGC CAT CTA TCC AAT CAG TCC AAT-3′ (reverse primer).

Chen R., Liang Y., Ip M.S., Zhang K.Y, & Mak J.C. (2020). Amelioration of Cigarette Smoke-Induced Mucus Hypersecretion and Viscosity by Dendrobium officinale Polysaccharides In Vitro and In Vivo. Oxidative Medicine and Cellular Longevity, 2020, 8217642.

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Quantitative RT-PCR for mRNA Quantification

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Well-differentiated HBECs were harvested for RNA extraction using MiniBEST Universal RNA extraction kit (Takara) and reverse transcription of RNA was conducted with EvoScript Universal cDNA Master kit (Roche). Quantitative RT-PCR assay was performed using SYBR Green Real-Time Master Mix (Applied Biosystems). All procedures were conducted following the manuals provided by the manufacturer. Relative quantity of mRNA was obtained by using the comparative Ct method and normalized by housekeeping genes such as human ribosomal protein S13 as previously described [31 (link)]. The primers used are listed in Additional File 1: Table S1.

Chen R., Hui K.P., Liang Y., Ng K.C., Nicholls J.M., Ip M.S., Peiris M., Chan M.C, & Mak J.C. (2023). SARS-CoV-2 infection aggravates cigarette smoke-exposed cell damage in primary human airway epithelia. Virology Journal, 20, 65.

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RT-qPCR Analysis of Differentially Expressed Genes

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Ten differentially expressed genes were selected for RT qPCR analysis (Table S7). Primers for selected genes were designed by Primer 3 software^{63 (link)} or taken from published sequences^{64 (link)–66 (link)}. cDNA was synthesized from 2 µg of purified RNA, using Super Script III Reverse Transcriptase (Thermo Fisher Scientific, Carlsbad, CA), as per manufacturer’s protocol. Each sample was analyzed in triplicate qPCR reactions. The final reaction volume of 10 µl contained 2 µl of cDNA, 8 µl of qPCR master mix (5 µl of SYBR Green Real-Time master mix (Applied Biosystems,Vilnius, Lithuania), 0.3 µl of each primer, 2.4 µl of DNA/RNA-free water). The samples were run on QuantStudio 5 Real-Time PCR System (Applied Biosystems). Standard curve calculation using four points of cDNA serial dilutions was used to estimate the PCR efficiency.

Arora R., Siddaraju N.K., Manjunatha S.S., Sudarshan S., Fairoze M.N., Kumar A., Chhabra P., Kaur M., Sreesujatha R.M., Ahlawat S, & Vijh R.K. (2021). Muscle transcriptome provides the first insight into the dynamics of gene expression with progression of age in sheep. Scientific Reports, 11, 22360.

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Quantitative PCR Analysis of P4HA1 mRNA

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All commercial assays used were performed according to the manufacturer's instructions. Total mRNA from cultured SW620 and HCT116 cells and tissue samples was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.),. Complementary DNA was synthesized from 2 µg total RNA using a Reverse Transcription kit (Takara Biotechnology Co., Ltd.) according to the manufacturers' instructions. Real-time quantitative PCR analyses were performed with SYBR-Green Real-Time Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following thermocycling conditions: Initial denaturation at 95°C, followed by 30 cycles at 95°C for 15 sec and 60°C for 1 min. The following primer pairs were used for qPCR: P4HA1 forward, 5′-AGGGGTTGCTGTGGATTACC-3′ and reverse, 5′-GTCATGTACTGTAGCTCGGC-3′; and GAPDH forward, 5′-GGGCTGCTTTTAACTCTGGT-3′ and reverse, 5′-TGGCAGGTTTTTCTAGACGG-3′. The Applied Biosystems 7500 Fast software version 1.4 (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to analyze the Cq values of different. Comparisons were made using the 2^−ΔΔCt method (19 (link)) and mRNAs normalized to an endogenous control GAPDH.

Zhang Q., Yin Y., Zhao H., Shi Y., Zhang W., Yang Z., Liu T., Huang Y, & Yu Z. (2020). P4HA1 regulates human colorectal cancer cells through HIF1α-mediated Wnt signaling. Oncology Letters, 21(2), 145.

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Quantification of miR-132 and miR-212

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Total RNAs were extracted from adrenal tissues, cultured rat ovarian granulosa cells, MLTC-1 Leydig cells or Y-1 adrenal cells using miRNeasy Isolation Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. RNA samples were reverse transcribed using the mercury LNA universal RT microRNA PCR kit (Exiqon Inc., Woburn, MA). Following cDNA synthesis, quantitative real-time PCR was performed on an ABI Prism 7900 HT system using SYBR® Green Real-time Master Mix (Invitrogen) and microRNA LNA PCR primer sets for miR-132 and miR-212 (Exiqon Inc.). Fold-changes of mRNA levels were determined after normalization to internal control, U6mRNA levels, a small nuclear gene, using the 2^−ΔΔCt method (Hu et al. 2013b (link)).

Hu Z., Shen W.J., Kraemer F.B, & Azhar S. (2017). Regulation of adrenal and ovarian steroidogenesis by miR-132. Journal of molecular endocrinology, 59(3), 269-283.

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Profiling miRNA expression via qRT-PCR

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All primers were provided from Bioneer, South Korea. MirPremier microRNA isolation kit was sourced from Sigma-Aldrich, USA. Mir-X miRNA First-Strand Synthesis kit and cDNA matermix were purchased from Takara bio inc, USA. SYBR® Green Real-Time Master Mix was from Invitrogen, UK.

Keikha R., Hashemi-Shahri S.M, & Jebali A. (2023). The miRNA neuroinflammatory biomarkers in COVID-19 patients with different severity of illness. Neurologia, 38(6), e41-e51.

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Profiling miRNA expression via qRT-PCR

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Keikha R., Hashemi-Shahri S.M, & Jebali A. (2021). The miRNA neuroinflammatory biomarkers in COVID-19 patients with different severity of illness. Neurologia (Barcelona, Spain), 38(6), e41-e51.

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Quantifying mRNA Expression in Y-1 Cells

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Total RNA from Y-1 cells transfected with ± miR-132 mimic or scrambled oligonucleotide and treated with ± Bt₂cAMP was extracted using a miRNeasy Mini Kit (Qiagen). To measure mRNA expression, total RNA (2 µg) from Y-1 cell samples was reverse transcribed using Superscript Reverse Transcriptase (Invitrogen) and oligo (dT) primers. The resulting complementary cDNA samples were used for qRT-PCR using SYBR® Green Real-time Master Mix (Invitrogen) and specific primers as listed in Table 1. qPCR was performed on an ABI Prism 7900 HT quantitative PCR system (Applied Biosystems). Target mRNA expression in each sample was normalized to the 36B4 signal. The 2^−ΔΔCt method was used to calculate relative mRNA expression levels.

Hu Z., Shen W.J., Kraemer F.B, & Azhar S. (2017). Regulation of adrenal and ovarian steroidogenesis by miR-132. Journal of molecular endocrinology, 59(3), 269-283.

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