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Superscript first strand synthesis system for reverse transcription pcr

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The Superscript First-Strand Synthesis System is a reverse transcription kit used to synthesize first-strand cDNA from RNA templates. It provides a simple and efficient method for performing reverse transcription–PCR.

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Lab products found in correlation

Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions) Rneasy lipid tissue kit, by Qiagen (4 mentions) 7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions) Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Iq sybr green supermix, by Bio-Rad (1 mentions) Rnase free dnase, by Thermo Fisher Scientific (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions) Purelink micro to midi kit, by Thermo Fisher Scientific (1 mentions) Lightcycler software 4, by Roche (1 mentions) Lightcycler faststart dna masterplus sybr green 1 kit, by Roche (1 mentions) Lightcycler 480, by Roche (1 mentions) Rnaiso plus, by Takara Bio (1 mentions) Rneasy kit, by Qiagen (1 mentions) Turbo dna free kit, by Thermo Fisher Scientific (1 mentions) Quantstudio 6 flex system, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions)

Close spelling variants of this product

SuperScript III First-Strand Synthesis System for reverse transcription-PCR SuperScript first-strand synthesis system for reverse transcription-PCR (RT-PCR) Superscript Reverse Transcription System First-Strand Synthesis System Reverse Transcription System

11 protocols using superscript first strand synthesis system for reverse transcription pcr

1

Transcriptional Profiling of Lipid-Related Genes

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Total RNA extracted from the liver and colon tissues was reverse-transcribed to cDNA using a SuperScript™ first-strand synthesis system for reverse transcription PCR (RT-PCR) (Invitrogen, United States). RT-PCR was performed on a LightCycler96 (Hangzhou Bioer Technology, Share-Holding Co.) using iQ SYBR Green Supermix (BIO-RAD, United States). The relative expression of lipid synthesis-related genes (Scd1, PPARγ [34 (link)]), lipid hydrolysis-related genes (Adrb3, Lipe, Pnpla2 [35 (link)]), lipid oxidation-related genes (Cpt2, Acox1, Ppargc1a [35 (link)], PPARα [36 (link)]), lipid transport gene (Fabp5 [37 (link)]), intestinal permeability-related genes (Occludin, ZO-1, Muc5 [38 (link)]), and inflammation-related genes (CD14 [39 (link)], TLR2, TLR4, NLRC4, and MCP-1 [40 (link)]) were adjusted with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. Relative quantification was calculated using the 2−∆∆Ct method. The primer sequences for RT-PCR can be found in Supplementary Table 1.
Wang R.R., Zhang L.F., Chen L.P., Wang J.Y., Zhang L., Xu Y.S., Yang P.L., Ji G, & Liu B.C. (2021). Structural and Functional Modulation of Gut Microbiota by Jiangzhi Granules during the Amelioration of Nonalcoholic Fatty Liver Disease. Oxidative Medicine and Cellular Longevity, 2021, 2234695.
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2

RNA Extraction and qRT-PCR Analysis

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Differentiated macrophages were harvested, and total RNA was extracted using TRIzol® (Thermo Fisher Scientific) according to the manufacturer’s instructions. The resulting RNA was then treated with RNase-free DNase (Ambion, California, USA) to remove any potential DNA contamination. After being reverse-transcribed to cDNA using the Super Script First Strand Synthesis System for reverse-transcription PCR (Invitrogen, Massachusetts, USA), triplicate cDNA aliquots were amplified with sequence-specific primers and SYBR green (Applied Biosystems by Life Technologies, California, USA) using 7500 Fast Real-Time PCR Systems (Applied Biosystems by Life Technologies). All reactions were repeated independently at least three times to ensure the reproducibility of the results. All primers were purchased from Sigma (Supplementary Table S1).
Inoue M., Niki M., Ozeki Y., Nagi S., Chadeka E.A., Yamaguchi T., Osada-Oka M., Ono K., Oda T., Mwende F., Kaneko Y., Matsumoto M., Kaneko S., Ichinose Y., Njenga S.M., Hamano S, & Matsumoto S. (2018). High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages. Scientific Reports, 8, 6736.
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3

Quantitative Analysis of Aquaporin-1 and GAP-43 Expression

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L4-6 DRGs (in control and sciatic nerve crush studies) were collected after euthanasia, total RNA was isolated by a PureLinkTM Micro-to-midi kit (Invitrogen), and cDNA was reverse-transcribed from mRNA with the Super-Script First Strand Synthesis System for reverse transcription-PCR (Invitrogen). Fluorescence-based quantitative real-time reverse transcription-PCR was carried out using the LightCycler 480 and with LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics). Primers were as follows: 5′-TGTATGCCTCTGGTCGTACC-3′ (sense) and 5′-CAGGTCCAGACGCAGGATG-3′ (antisense) for GAPDH; 5′-CTCAACTACATGGTCTACATGTTCCA-3′ (sense) and 5′- CCATTTCGGCCTTGACTGT-3′ (antisense) for AQP1, 5′-CAGGAAAGATCCCAAGTCCA-3′ (sense) and 5′-GAACGGAACATTGCACACAC-3′ (antisense) for GAP-43. Data were analyzed by LightCycler software 4.0 (Roche Diagnostics) and reported as calibrated ratios normalized to GAPDH. Data were averaged from six mice.
Zhang H, & Verkman A.S. (2015). AQUAPORIN-1 WATER PERMEABILITY AS A NOVEL DETERMINANT OF AXONAL REGENERATION IN DORSAL ROOT GANGLION NEURONS. Experimental neurology, 265, 152-159.
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4

Quantifying Gene Expression in HaCaT Cells

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Total cellular RNA was isolated from HaCaT cells according to TRI reagent-specific instruction (RNA iso Plus, Takara Bio. Inc., Shiga, Japan). cDNA was synthesized using reagent-specific instruction of Superscript First Strand synthesis system for reverse transcription-PCR (Invitrogen, Carlsbad, CA, USA) from isolated RNAs. Primers used for real-time PCR were as follows: COX-1 forward, 5’-CCTCAT GTTTGCCTTCTTTGC-3’ and reverse, 5’-GGC GGGTACATTTCTCCATC-3’ COX-2 forward, 5’-GATCTA CCCTCCTCAA-3’ and reverse, 5’-GAACAACTGC TCATCAC-3’ MRP4 forward, 5’-AACCTCTAACCGACATTCCTG-3’ and reverse, 5’-TCAACATA TTACAGCCACCATC-3’ human PGT forward, 5’-GGATGCTGTTTGGAGGAATCCTCA-3’ and reverse, 5’-GCACGATCCTGTC TTTGCTGAA-3’ and β-actin forward, 5’-GACTATGACTTAGTTGCGTTA-3’ and reverse, 5’-GTTGAACTCTCTACATAC TTCCG-3’. PCR reaction mixture contained 4 µL of diluted cDNA (1:5), 10 pmole of each forward and reverse primer, 4 mM MgCl2, and 4 µL of Fast Starter Mix buffer (dNTPs, SYBR Green dye and Tag polymerase).
, & Karna S. (2017). In-vitro Wound Healing Effect of 15-Hydroxyprostaglandin Dehydrogenase Inhibitor from Plant. Pharmacognosy Magazine, 13(Suppl 1), S122-S126.
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5

Transcriptome Analysis using qRT-PCR

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RNA extractions from tissues were performed using the RNeasy Lipid Tissue Kit (Qiagen) While RNA extraction from cells utilized the RNeasy Kit, we used the Superscript First-Strand Synthesis System for reverse transcription-PCR (Invitrogen) with a 3:1 mixture of random hexamers/oligo dT primers for reverse transcription. Real-time PCR amplification was performed on samples in triplicate with Power SYBR Green PCR Master Mix (Applied Biosystems) using the Applied Biosystems 7900HT Fast real-time PCR System and quantified using an internal standard curve and Arbp as the control gene. The sequences of all primers used in this study are listed in Supplementary Table 2.
Reilly S.M., Ahmadian M., Zamarron B.F., Chang L., Uhm M., Poirier B., Peng X., Krause D.M., Korytnaya E., Neidert A., Liddle C., Yu R.T., Lumeng C.N., Oral E.A., Downes M., Evans R.M, & Saltiel A.R. (2015). A subcutaneous adipose tissue–liver signalling axis controls hepatic gluconeogenesis. Nature Communications, 6, 6047.
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6

Quantitative Gene Expression Analysis

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RNA extractions from tissues were performed using the RNeasy Lipid Tissue Kit (Qiagen) While RNA extraction from cells utilized the RNeasy Kit, we used the Superscript First-Strand Synthesis System for reverse transcription–PCR (Invitrogen) with a 3:1 mixture of random hexamers/oligo dT primers for reverse transcription. Real-time PCR amplification was performed on samples in triplicate with Power SYBR Green PCR Master Mix (Applied Biosystems) using the Applied Biosystems 7900HT Fast real-time PCR System and quantified using an internal standard curve and Arbp as the control gene. The sequences of all primers used in this study are listed in Supplementary Table 2.
Reilly S.M., Ahmadian M., Zamarron B.F., Chang L., Uhm M., Poirier B., Peng X., Krause D.M., Korytnaya E., Neidert A., Liddle C., Yu R.T., Lumeng C.N., Oral E.A., Downes M., Evans R.M, & Saltiel A.R. (2015). A subcutaneous adipose tissue–liver signalling axis controls hepatic gluconeogenesis. Nature Communications, 6, 6047.
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7

Quantification of Cellular mRNA Levels

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To quantify mRNA levels of various genes, RNA samples were isolated from cells using the RNeasy Kit (Qiagen), and cDNA were prepared using the Superscript First Strand Synthesis System for reverse transcription PCR (RT-PCR) (Invitrogen). Real time PCR analysis was done using a custom plate from RT2 Custom Profiler PCRArray CAPA9696-24 (SABiosciences) and analyzed using the Web-based PCR Array Data Analysis. Fold changes in mRNA levels were calculated after normalization to housekeeping genes [β-Actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), 60 S acidic ribosomal protein P0 (RPLP0)].
Mahadev V., Starr R., Wright S.L., Martinez C., Jensen M.C., Barish M.E., Forman S.J, & Brown C.E. (2014). Cytokine Induction of VCAM-1 but Not IL13Rα2 on Glioma Cells: A Tale of Two Antibodies. PLoS ONE, 9(5), e95123.
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8

Quantitative RT-PCR Gene Expression Analysis

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Total RNA was extracted by an RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. Isolated RNA was treated with deoxyribonuclease to remove DNA (Turbo DNA-free kit, Ambion). One microgram of isolated total RNA was then immediately reverse-transcribed into complementary DNA using the SuperScript First-Strand Synthesis System for reverse transcription PCR (Invitrogen). qPCR was performed with a QuantStudio 6 Flex system (Applied Biosystem) using SYBR green qPCR mastermix in a 384-well optical plate. PCR cycling consists of 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. A melt curve was conducted to determine the specificity of PCR amplification. Gapdh or Sdha served as an internal control to normalize data.
Xiao M.F., Roh S.E., Zhou J., Chien C.C., Lucey B.P., Craig M.T., Hayes L.N., Coughlin J.M., Leweke F.M., Jia M., Xu D., Zhou W., Conover Talbot C J.r., Arnold D.B., Staley M., Jiang C., Reti I.M., Sawa A., Pelkey K.A., McBain C.J., Savonenko A, & Worley P.F. (2021). A biomarker-authenticated model of schizophrenia implicating NPTX2 loss of function. Science Advances, 7(48), eabf6935.
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9

RNA Extraction and qPCR Analysis of WAT

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RNA extractions from inguinal WAT were performed using the RNeasy Lipid Tissue Kit (Qiagen). We used the Superscript First-Strand Synthesis System for reverse transcription–PCR (Invitrogen) with a 3:1 mixture of random hexamers/oligo dT primers for reverse transcription. Real-time PCR amplification was performed on samples in triplicate with Power SYBR Green PCR Master Mix (Applied Biosystems) using the Applied Biosystems QuantStudio5 real-time PCR System and quantified using an internal standard curve with Arbp as the control gene. The sequences of all primers used in this study are listed in Supplementary Table 1.
Reilly S.M., Hung C.W., Ahmadian M., Zhao P., Keinan O., Gomez A.V., DeLuca J.H., Dadpey B., Lu D., Zaid J., Poirer B., Peng X., Yu R.T., Downes M., Liddle C., Evans R.M., Murphy A.N, & Saltiel A.R. (2020). Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3. Nature metabolism, 2(7), 620-634.
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10

RNA Extraction and qPCR Analysis of WAT

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RNA extractions from inguinal WAT were performed using the RNeasy Lipid Tissue Kit (Qiagen). We used the Superscript First-Strand Synthesis System for reverse transcription–PCR (Invitrogen) with a 3:1 mixture of random hexamers/oligo dT primers for reverse transcription. Real-time PCR amplification was performed on samples in triplicate with Power SYBR Green PCR Master Mix (Applied Biosystems) using the Applied Biosystems QuantStudio5 real-time PCR System and quantified using an internal standard curve with Arbp as the control gene. The sequences of all primers used in this study are listed in Supplementary Table 1.
Reilly S.M., Hung C.W., Ahmadian M., Zhao P., Keinan O., Gomez A.V., DeLuca J.H., Dadpey B., Lu D., Zaid J., Poirer B., Peng X., Yu R.T., Downes M., Liddle C., Evans R.M., Murphy A.N, & Saltiel A.R. (2020). Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3. Nature metabolism, 2(7), 620-634.
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