Quanta qscript cdna supermix

Manufactured by Quanta Biosciences

Quanta qScript cDNA SuperMix is a ready-to-use reagent for the reverse transcription of RNA to cDNA. It contains all the necessary components, including reverse transcriptase, RNase inhibitor, and buffer, to efficiently convert RNA into cDNA.

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Lab products found in correlation

Perfecta sybr green fastmix for iq, by Quanta Biosciences (3 mentions) Cfx96, by Bio-Rad (3 mentions) Nanodrop spectrophotometer, by Isogen Life Science (2 mentions) Sybr green, by Thermo Fisher Scientific (2 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Qiazol lysis reagent, by Qiagen (2 mentions) Rneasy column, by Qiagen (2 mentions) Perfecta sybr green fastmix, by Quanta Biosciences (1 mentions) Mirneasy micro kit, by Qiagen (1 mentions) Trizol, by Qiagen (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Rneasy plant mini kit, by Qiagen (1 mentions) Icycler, by Bio-Rad (1 mentions)

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QScript cDNA SuperMix Quanta cDNA SuperMix Quanta qScript QScript Supermix

8 protocols using quanta qscript cdna supermix

Estrogen Regulation of PCNA Expression

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Cells were grown in six-well plates at a density of 5 × 10⁵ cells/mL in 1 % charcoal-stripped FBS media (Invitrogen) for 24 h prior to the start of each experiment. Cells were incubated with vehicle (dimethyl sulfoxide, DMSO, final 0.1 %) or E2 (0, 1, 10, 50, or 100 nM) for 48 h. RNA was extracted from cells using the Promega SV Total RNA Isolation System for Tissues and reverse transcribed (0.2 μg) using the Quanta qScript cDNA supermix (Quanta Biosciences). cDNA was diluted (1:10) and amplified using the Perfecta SYBR Green FastMix (Quanta Biosciences). Groups were normalized to vehicle using the delta delta Ct method (ddCt). Glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA abundance was used to control for DNA template concentrations. Primers for proliferating cell nuclear antigen (PCNA) [17 (link)], forward 5′-CTAGCCATGGGCGTGAAC-3′ reverse 5′-GAATACTAGTGCTAAGGTGTCTGCAT-3′ and GAPDH, forward 5′-GCCAAAAGGGTCATCATCTC-3′ reverse 5′-GGCCATCCACAGTCTTCT-3′ were utilized for this study.

Thatcher S.E., Zhang X., Woody S., Wang Y., Alsiraj Y., Charnigo R., Daugherty A, & Cassis L.A. (2015). Exogenous 17-β estradiol administration blunts progression of established angiotensin II-induced abdominal aortic aneurysms in female ovariectomized mice. Biology of Sex Differences, 6, 12.

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Knockdown of BAF150 and BAF170 Regulates miR-9 Expression

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Neurospheres were suspended into single cells suspension and plated in 24-well cell culture plates. Accell siRNA reagents (GE Dharmacon, Lafayette, CO) were used to knockdown BAF150 and BAF170. Accell Mouse Smarcc1 (E-044249) siRNA and Accell Mouse Smarcc2 (E-042842) siRNA – SMARTpool were delivered together to culture wells at a final concentration of 1µM each. Accell Non-targeting Pool (siRNA control, D-001910-10) was delivered to control wells at 2µM final concentration and Accell Cyclophilin B Pool (D-001920-20) was used to assess the transfection efficiency. After 72 hours of transfection, RNA was isolated by the Qiagen miRNeasy micro kit (Qiagen). The Quanta qScript cDNA Supermix (Quanta Biosciences, Beverly, MA) was used for first strand cDNA synthesis. Knock down of BAF150 and BAF170 was confirmed using qRT-PCR. After knockdown, miR-9 expression levels were analyzed using the Exiqon miRCURY LNA Universal RT microRNA cDNA synthesis kit (Exiqon A/S, Vedbaek Denmark) was used to synthesize cDNA qRT-PCR, which was subsequently performed using a commercial miR-9 primer set (Exiqon, #202240) with the small nuclear RNA, U6 (Exiqon, #203907), as a loading control

Burrowes S.G., Salem N.A., Tseng A.M., Balaraman S., Pinson M.R., Garcia C, & Miranda R.C. (2017). The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus. Alcohol (Fayetteville, N.Y.), 60, 149-158.

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Quantitative PCR Gene Expression Analysis

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Cell lysis was performed by using Qiazol Lysis reagent (Qiagen). RNA was extracted using the RNeasy Mini Kit (Qiagen). RNA concentration and quality were determined with a Nanodrop spectrophotometer (Isogen Life Science). Complementary DNA (cDNA) synthesis was performed by using the Quanta qScript cDNA SuperMix (Quanta Biosciences) per manufacturer instructions. qPCR was conducted on the StepOnePlus Real-Time PCR System (Applied Biosystems) using a SYBR green mix containing 1× SYBR green (Applied Biosystems), 0.3 μM primers (Integrated DNA Technologies), 12.5 ng cDNA, and nuclease-free water. The comparative Ct method was used to quantify gene expression. Data were normalized to the most stable reference genes cyclin A and hypoxanthine phosphoribosyltransferase 1. Primer sequences are available in SI Appendix, Table S1.

Dierckx T., Vanherle S., Haidar M., Grajchen E., Mingneau F., Gervois P., Wolfs E., Bylemans D., Voet A., Nguyen T., Hamad I., Kleinewietfeld M., Bogie J.F, & Hendriks J.J. (2022). Phloretin enhances remyelination by stimulating oligodendrocyte precursor cell differentiation. Proceedings of the National Academy of Sciences of the United States of America, 119(46), e2120393119.

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RNA-Seq Complementary DNA Synthesis

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RNA was isolated as described above for RNA‐Seq. First‐strand complementary DNA (cDNA) synthesis was performed using Quanta qScript cDNA SuperMix (Quanta BioSciences, https://www.quantabio.com), following the manufacturer’s instructions. qPCR was performed on a BioRad CFX96 using PerfeCTa sybr Green FastMix for iQ (Quanta BioSciences) and primers listed in Table S5. Data analyses were carried out as described previously (Krizek and Eaddy, 2012). Two biological replicates were analyzed for each experiment.

Krizek B.A., Blakley I.C., Ho Y., Freese N, & Loraine A.E. (2020). The Arabidopsis transcription factor AINTEGUMENTA orchestrates patterning genes and auxin signaling in the establishment of floral growth and form. The Plant Journal, 103(2), 752-768.

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Transcriptional Regulation of Arabidopsis Genes

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35S:AlcR/AlcA:AIL6-amiR ant-4 and 35S:ALCR/AlcA:ANT-IR plants were mock treated or treated with ethanol vapor by placing 2 ml of water or 2 ml of 100% ethanol in 2 ml centrifuge tubes in half of the pots in a tray. The tray was covered with a plastic dome. Inflorescences were collected at the end of an 8 h (35S:AlcR/AlcA:AIL6-amiR ant-4) or 24 h (35S:ALCR/AlcA:ANT-IR) treatment. RNA was isolated using an RNeasy Plant Mini Kit (Qiagen) or TRIzol (Life Technologies). Samples isolated with TRIzol were further purified on an RNeasy column (Qiagen) and treated with DNase while on the column. First-strand cDNA synthesis was performed using Quanta qScript cDNA SuperMix (Quanta BioSciences) following the manufacturer’s instructions. Quantitative PCR (qPCR) was performed on a BioRad CFX96 or CFX Connect real-time PCR system using PerfeCTa SYBR Green FastMix for iQ (Quanta BioSciences) and primers listed in Supplementary Table S1. Data analyses were carried out as described previously (Krizek and Eaddy, 2012 (link)). At least two biological replicates were analyzed for each experiment.

Krizek B.A., Bantle A.T., Heflin J.M., Han H., Freese N.H, & Loraine A.E. (2021). AINTEGUMENTA and AINTEGUMENTA-LIKE6 directly regulate floral homeotic, growth, and vascular development genes in young Arabidopsis flowers. Journal of Experimental Botany, 72(15), 5478-5493.

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RNA Extraction and Quantification Protocol

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RNA was extracted from inflorescences using Trizol following the manufacturer’s instructions with cleanup on an RNeasy column (Qiagen). The RNA was DNased while on the column. First strand cDNA synthesis was performed using Quanta qScript cDNA SuperMix (Quanta BioSciences) following the manufacturer’s instructions. RT-PCR and real-time PCR was performed using the primers listed in Supplementary Table S1 at JXB online. The RT-PCR experiment usd the following PCR conditions: 40 cycles of 92 °C for 30 s, 55 °C for 30 s, and 72 °C for 2min followed by 1 cycle of 72 °C for 5min. Real-time PCR was performed on a BioRad iCycler as described previously (Krizek and Eaddy, 2012 (link)).

, & Krizek B.A. (2015). AINTEGUMENTA-LIKE genes have partly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flower development. Journal of Experimental Botany, 66(15), 4537-4549.

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Quantitative Real-Time PCR Protocol

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RNA was isolated as described above for RNA-Seq. First strand complementary DNA (cDNA) synthesis was performed using Quanta qScript cDNA SuperMix (Quanta BioSciences) following the manufacturer’s instructions. qPCR was performed on a BioRad CFX96 using PerfeCTa SYBR Green FastMix for iQ (Quanta BioSciences) and primers listed in Table S5. Data analyses were carried out as described previously (Krizek and Eaddy 2012 (link)). Two biological replicates were analyzed for each experiment.

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Phloretin-LPS Induced Gene Expression

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Cells were pretreated with phloretin (50 μM) for 20 h and LPS stimulated (100 ng/ml) for 6 h. Lysis was performed by using Qiazol Lysis reagent (Qiagen). RNA was extracted using the RNeasy mini kit (Qiagen). RNA concentration and quality were determined with a Nanodrop spectrophotometer (Isogen Life Science). cDNA synthesis was conducted using the Quanta qScript cDNA SuperMix (Quanta Biosciences) per the manufacturer’s instructions. qPCR was performed on a StepOnePlus™ Real-Time PCR system (Applied biosystems) using a SYBR green mix containing 1× SYBR green (Applied Biosystems), 0.3 μM primers (Integrated DNA Technologies), 12.5 ng cDNA and nuclease-free water. The comparative Ct method was used to quantify gene expression. Data were normalized to the most stable reference genes cyclin A and hypoxanthine phosphoribosyltransferase 1. Primer sequences are available on request.

Dierckx T., Haidar M., Grajchen E., Wouters E., Vanherle S., Loix M., Boeykens A., Bylemans D., Hardonnière K., Kerdine-Römer S., Bogie J.F, & Hendriks J.J. (2021). Phloretin suppresses neuroinflammation by autophagy-mediated Nrf2 activation in macrophages. Journal of Neuroinflammation, 18, 148.

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