Sybr green master mix kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Belgium, Japan, China

The SYBR Green Master Mix kit is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I dye, DNA polymerase, dNTPs, and buffer components.

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145 protocols using sybr green master mix kit

Quantitative PCR Analysis of Hippo Pathway

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Real-time PCR assays were performed on the StepOnePlus (Thermo Fisher) using the TaqMan gene expression assay kit (Applied Biosystems ® ). The Hippo pathway probes used are described in Table S3.
As the TaqMan probe (Hs00371735_m1) did not detect the YAP1 gene, we used the Sybr green master mix kit (Thermo Fisher) and the primer pair sequence employed for RNA quanti cation described in Table S4. The Sybr green master mix kit (Thermo Fisher) was also used to analyze pro-and anti-apoptotic gene expression. The oligonucleotide sequences employed in ampli cation of the target genes are described in Table S4. The gene expression results were reported as relative expression unit (REU = 10000 / 2DCt) for patients or Fold Change for cell line. The DCT = CT of the target gene -geometric mean of the reference genes CTs. REU = 10000 / 2deltaCt.

Cacemiro M.D.C., Cominal J.G., Pereira L.M., Berzoti-Coelho M.G., Berbel G.M., Baroni L., Malta T., Tognon R., Nunes N.S., Souto E.X., de Figueiredo-Pontes L.L., Yatsuda A.P, & de Castro F.A. (2022). Hippo pathway-related genes expression is deregulated in myeloproliferative neoplasms. Medical oncology (Northwood, London, England), 39(5).

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Quantifying Gene Expression in Glioma Samples

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Total RNA was extracted from glioma specimens or glioma cells using TRIzol reagent (Invitrogen). Real-time quantitative PCR analysis was performed using SYBR Green Master Mix Kit on Thermo Fisher connect Real-Time PCR platform. For relative quantification, 2^−ΔΔCT was calculated and used as an indication of the relative expression levels, which was calculated by subtracting CT values of the control gene from the CT values of miR-141, HOTAIR, SKA2 and DNMT1. Real time PCR was carried out under a standard protocol using the following primers(Supplementary File).

Bian E.B., Ma C.C., He X.J., Wang C., Zong G., Wang H.L, & Zhao B. (2016). Epigenetic modification of miR-141 regulates SKA2 by an endogenous ‘sponge’ HOTAIR in glioma. Oncotarget, 7(21), 30610-30625.

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Quantitative RT-PCR Analysis of Monocyte Genes

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Total RNA from one million of cells suspended in Trizol was isolated on silica columns (A&A Biotechnology) followed by the cDNA synthesis (Roche). Quantitative RT-PCR was performed using a fluorogenic SYBR Green Master Mix kit (Thermo Fisher) and a Mx3005P QPCR System (Agilent). Data were analyzed using the comparative Ct method [19 (link)]. Sequences of key genes involved in monocyte/macrophage biology were obtained from Primer Bank [20 (link)] (S1 Table). Ten different genes were tested as possible housekeeping genes. Finally, results were normalized to eukaryotic elongation factor 2 (eef2) housekeeping gene. Expression of other genes existing in the literature as possible housekeeping genes (Actb, B2m, Gusb, Hprt, Hsp90ab1, Ldha16b, Nono, Ppia, Rpl13a and Tbp) was less stable (S1 Fig). Gene expression was analyzed in RAW 264.7 cells of the following passage no.: 5, 10, 15, 20, 30 and 50. This analysis has been performed on the cells obtained from two various cell sources in triplicate.

Taciak B., Białasek M., Braniewska A., Sas Z., Sawicka P., Kiraga Ł., Rygiel T, & Król M. (2018). Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages. PLoS ONE, 13(6), e0198943.

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Quantitative RT-PCR Analysis of Inflammatory Genes

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RNA was extracted using the TRIzol method (Invitrogen) and reverse transcribed with Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using the SYBR Green Master mix kit (Thermo Fisher). Primer sequences were as follows:
Rpl32 (F) ACATCGGTTATGGGAGCAAC; Rpl32 (R) TCCAGCTCCTTGACATTGT; Il1b (F) CAAGCTTCCTTGTGCAAGTG; Il1b (R) AGGTGGCATTTCACAGTTGA; Il10 (F) CCTGGGTGAGAAGCTGAAGA; Il10 (R) GCTCCACTGCCTTGCTCTTA; Ifng (F) TGCCAAGTTTGAGGTCAACA; Ifng (R) GAATCAGCAGCGACTCCTTT; Il6 (F) GTTCTCTGGGAAATCGTGGA; Il6 (R) GCAAGTGCATCATCGTTGTT; Il17a (F) ATCCCTCAAAGCTCAGCGTGTC; Il17a (R) GGGTCTTCATTGCGGTGGAGAG; Il12a (F) CCTCAGTTTGGCCAGGGTC; Il12a (R) CAGGTTTCGGGACTGGCTAAG; Il12b (F) ATGTGTCCTCAGAAGCTAACC; Il12b (R) CTAGGATCGGACCCTGCAGGGAAC; Tnfa (F) GCCTCCCTCTCATCAGTTCTA; Tnfa (R) GCTACGACGTGGGCTACAG; Egln1 (F) AGGCTATGTCCGTCACGTTG; Egln1 (R) TACCTCCACTTACCTTGGCG; Hif1a (F) CATCAGTTGCCACTTCCCCA; Hif1a (R) GGCATCCAGAAGTTTTCTCACAC; Epas1 (F) ACGGAGGTCTTCTATGAGTTGGC; Epas1 (R) GTTATCCATTTGCTGGTCGGC.

Ajouaou Y., Azouz A., Taquin A., Denanglaire S., Hussein H., Krayem M., Andris F., Moser M., Goriely S, & Leo O. (2022). The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells. eLife, 11, e70555.

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Extraction and Quantification of Total RNA

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Total RNA was extracted with TRIzol (cat# 15596026, Invitrogen) and reversely transcribed using TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix (cat# AH311-03, TransGen Biotech). The qRT-PCR was completed using the SYBR Green Master Mix kit (cat# A25742, Thermo Fisher Scientific), and the assay was conducted with the QuantStudio5 Real-Time PCR System (Life Technologies). The primers are listed in Supplementary file 2.

Geng G., Liu J., Xu C., Pei Y., Chen L., Mu C., Wang D., Gao J., Li Y., Liang J., Zhao T., Zhang C., Zhou J., Chen Q., Zhu Y, & Shi L. (2021). Receptor-mediated mitophagy regulates EPO production and protects against renal anemia. eLife, 10, e64480.

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Quantitative Real-Time PCR Analysis of Oxidative Stress Genes

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According to the instructions of manufacturer, total RNA from MG-63 and U2OS cells was extracted using TRIzol reagent (Sigma-Aldrich). Total RNA was reverse-transcribed to CDNA using a Reverse Transcriptase kit (Thermo Scientific, Waltham, MA, USA). RT-qPCR was performed with SYBR® Green Master Mix Kit (Thermo Scientific) and the Mastercycler ep realplex detection system (Eppendorf, Hamburg, Germany). The relative mRNA expression was normalized to β-actin by using the 2^−ΔΔCt method. Primer sequences are shown in Table 1.

Table 1

Primers for qRT-PCR in this study

Gene	Sequence from 5’-3’
FANCD2 Forward	ACATACCTCGACTCATTGTCAGT
FANCD2 Reverse	TCGGAGGCTTGAAAGGACATC
FTH1 Forward	CGCCAGAACTACCACCAG
FTH1 Reverse	TTCAAAGCCACATCATCG
GPX4 Forward	GAAGCAGGAGCCAGGGAGT
GPX4 Reverse	ACGCAGCCGTTCTTGTCG
COX2 Forward	TGGAGCACCATTCTCCTTGAAAGGACTTAT
COX2 Reverse	GACTGTTTTAATGAGCTCTGGATCTGGAAC
GAPDH Forward	GAATTCATGTTTGAGACCTTCAA
GAPDH Reverse	CCGGATCCATCTCTTGCTCGAAGTCCA

Li X, & Liu J. (2023). FANCD2 inhibits ferroptosis by regulating the JAK2/STAT3 pathway in osteosarcoma. BMC Cancer, 23, 179.

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RT-qPCR Analysis of Gene Expression in hBMSCs

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The total RNA was extracted from hBMSCs and blood using the TRIzol reagent (Invitrogen, CA, USA). Subsequently, the PrimeScript RT reagent kit (Thermo Fisher Scientific, Inc) was used to reverse transcription of the RNA into the complementary DNA. RT-qPCR was subsequently performed on the Applied Biosystems Prism 7900 system (Thermo Fisher Scientific, Inc) in accordance with the instructions of the SYBR‑Green Master Mix kit (Thermo Fisher Scientific, Inc). The used primers were listed in Table 1.Table 1

Quantitative Polymerase Chain Reaction Primers for Reverse Transcription

Primer	Forward (5’‑3’)	Reverse (5’‑3’)
NEAT1	TGGCTAGCTCAGGGCTTCAG	TCTCCTTGCCAAGCTTCCTTC
miR-339-5p	CGCTCTCCCTGTCCTCCA	GCACACGTGAGCTCCTGG
SPI1	ATGAAGGACAGCATCTGGTGG	TTCACCTTCTTGACCTCGCCC
RUNX2	TGGTTACTGTCATGGCGGGTA	TCTCAGATCGTTGAACCTTGCTA
OSX	GAAGAAGCTCACTATGGCTC	GAAAAGCCAGTTGCAGACGA
ALPL	GAAAAGCCAGTTGCAGACGA	GTGGAACATC-GGTCCGGGTA
β‑actin	TCACCCACACTGTGCCCATCTACGA	CAGCGGAACCGCTCATTGCCAATGG
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

Zhu D., Zhu Z, & Qi H. (2023). NEAT1/microRNA 339-5p/SPI1 Axis Feedback Loop Contributes to Osteogenic Differentiation in Acute Suppurative Osteomyelitis in Children. Journal of Inflammation Research, 16, 2675-2687.

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Quantitative Analysis of miRNA Expression

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Total RNA was extracted from MLTC-1 cells according to the manufacturer’s specification using a Trizol kit (Thermo Fisher Scientific). Then, cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to synthesize cDNA. After that, PCR was performed by using SYBR Green master mix kit (Thermo Fisher Scientific). The qPCR conditions were as follows: 94°C for 5 mins; and 35 cycles of 95°C for 30 s, 60°C for 30 s and 75°C for 45 mins. The sequences of primers are as follows: miR-34a: Forward: 5’-ACAGTCTCATGCCAGGAAAGC-3’, Reverse: 5’-GCACATTGATGATGCACAGGC-3’; miR-15a: Forward: 5’-GCTAGCAGCACATAATGGTTTGTG-3’, Reverse: 5’-GTGCAGGGTCCGAGGTATTC-3’; miR-184: Forward: 5’-TACGACTATGTGACCTGCCTG-3’, Reverse: 5’- TGGTTCAACTCTCCTTTCCA-3’; miR-130a Forward: 5’-GGGGTACCGCTTACCCACATCATA-3’, Reverse: 5’-GAAGATCTTACCACCATGGATCGTC-3’; miR-26a: Forward: 5’- ACACTCCAGCTGGGTTCAAGTAATCCAGGA-3’, Reverse: 5’-TGGTGTCGTGGAGTCG-3’; GSK3β: Forward: 5′-CAAAGCAGCTGGTCCGAGG-3′, Reverse:5′-TCCACCAACTGATCCACACCAC-3’, U6: Forward: 5’- CGCTTCGGCAGCACATATAC-3’, Reverse: 5’-AAATATGGAACGCTTCACGA-3’. The relative levels of miR-34a, miR-15a, miR-184, miR-130a, miR-26a and GSK3B were normalized to the level of U6. The 2^−ΔΔCt method was used to analyze the data.26 (link)

Liang H., Zhang S, & Li Z. (2019). Ginsenoside Rg3 protects mouse leydig cells against triptolide by downregulation of miR-26a. Drug Design, Development and Therapy, 13, 2057-2066.

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Hippocampal RNA Extraction and qPCR Analysis

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Total RNA was extracted from the hippocampus using TRIzol reagent (TaKaRa Bio, Dalian, China) according to the manufacturer's instructions. RNA was reverse transcribed to cDNA by using a reverse transcriptase kit (TaKaRa Bio, Dalian, China) on a Stratagene Robocycler Gradient 96 Thermal Cycler (Stratagene, California, USA). Real-time PCR assays were performed using a SYBR green Master Mix kit (Thermo Scientific, California, USA; Cat. No. #K0251) on an Mx3005 system (Stratagene). The following primers were used in this study: β-actin, (forward) 5′-GCA GGA GTA CGA TGA GTC CG-3′ and (reverse) 5′-ACG CAG CTC AGT AAC AGT CC-3′; TNF-α, (forward) 5′-GAC ACC ATG AGC ACG GAA AGC A-3′, and (reverse) 5′-CGC CAC GAG CAG GAA TGA GAA G-3′. The mRNA expression levels of the target gene were normalized to those of β-actin. Real-time PCR was done 6 weeks after surgery.

Cai L., He Q., Lu Y., Hu Y., Chen W., Wei L, & Hu Y. (2019). Comorbidity of Pain and Depression in a Lumbar Disc Herniation Model: Biochemical Alterations and the Effects of Fluoxetine. Frontiers in Neurology, 10, 1022.

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Quantitative RT-PCR Analysis of Gene Expression

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qRT-PCR was performed using SYBR Green master mix kit from Thermo Fisher Scientific (Waltham, MA, USA). PCR was performed using 7500 Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used as the endogenous control. The fold change of target gene was calculated using the 2^−ΔΔCt method.

Du Y., Zhang W., Du B., Zang S., Wang X., Mao X, & Hu Z. (2018). TRIM32 overexpression improves chemoresistance through regulation of mitochondrial function in non-small-cell lung cancers. OncoTargets and therapy, 11, 7841-7852.

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