Superscript first strand cdna synthesis system for rt pcr

Manufactured by Thermo Fisher Scientific

The Superscript First-Strand cDNA Synthesis system is a reagent kit for reverse transcription of RNA into complementary DNA (cDNA) for use in reverse transcription polymerase chain reaction (RT-PCR) applications. The kit includes reagents necessary for the conversion of RNA to first-strand cDNA.

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SuperScript First-Strand Synthesis System for RT-PCR (305 citations) First-Strand Synthesis System (55 citations) RT-PCR system (41 citations) SuperScript RT-PCR system (20 citations) First-Strand cDNA Synthesis System (14 citations) CDNA synthesis system (7 citations) SuperScript III First-Strand cDNA synthesis system for RT-PCR (6 citations) SuperScript First-Strand System for RT-PCR (5 citations) Superscript First Strand RT-PCR system (3 citations) SuperScript III Transcriptor First Strand cDNA Synthesis System for RT-PCR (2 citations)

5 protocols using superscript first strand cdna synthesis system for rt pcr

Organ of Corti RNA Expression Analysis

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Mice were euthanized by CO₂ inhalation, then decapitated, ears removed by gross dissection, placed in ice-cold HBSS, and then microdissected for the organ of Corti. Organ of Corti RNA was isolated using the RNAqueous-Micro RNA Isolation Kit (Ambion, Austin, TX, USA). Isolated RNA was treated with DNase I before cDNA synthesis. cDNA was generated using Superscript First-Strand cDNA Synthesis system for RT-PCR (Invitrogen) with random primers.
Relative expression levels were assayed utilizing TaqMan Gene Expression Master Mix and TaqMan probes (Applied Biosystems, Foster City, CA, USA) for Cflar, Bcl2l1, Bcl2l11, Cdkn1a, Hspa1a, 18S, GAPDH, and Actb. Reactions were run in triplicate in an Eppendorf Realplex² Mastercycler System (Hauppauge, NY, USA). The level of 18S, GAPDH, and Actb were used as internal controls and were run as a multiplex reaction with each assayed gene. The difference in C_T between the assayed gene and 18S, GAPDH, and Actb for any given sample was defined as ΔC_T(X). The difference in ΔC_T(x) between two samples was defined as ΔΔC_T(X), which represents a relative difference in expression of the assayed gene. The fold change of the assayed gene relative to 18S, GAPDH, and Actb was defined as 2^−ΔΔCT.^{41 (link)} DataAssist software (Applied Biosystems) was used for statistical analysis and to confirm ΔC_T(X) calculation.

Layman W.S., Williams D.M., Dearman J.A., Sauceda M.A, & Zuo J. (2015). Histone deacetylase inhibition protects hearing against acute ototoxicity by activating the Nf-κB pathway. Cell Death Discovery, 1, 15012-.

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Quantitative RT-PCR Analysis of Cochlear Gene Expression

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Mice were euthanized by CO₂ inhalation, then decapitated, ears removed by gross dissection, placed in ice-cold HBSS, and then microdissected for the organ of Corti. Organ of Corti RNA was isolated using the RNAqueous-Micro RNA Isolation Kit (Ambion, Austin, TX, USA). Isolated RNA was treated with DNase I before cDNA synthesis. cDNA was generated using Superscript First-Strand cDNA Synthesis system for RT-PCR (Invitrogen) with random primers.
Relative expression levels were assayed utilizing TaqMan Gene Expression Master Mix and TaqMan probes (Applied Biosystems, Foster City, CA, USA) for Cflar, Bcl2l1, Bcl2l11, Cdkn1a, Hspa1a, 18S, GAPDH, and Actb. Reactions were run in triplicate in an Eppendorf Realplex^{2 (link)} Mastercycler System (Hauppauge, NY, USA). The level of 18S, GAPDH, and Actb were used as internal controls and were run as a multiplex reaction with each assayed gene. The difference in C_T between the assayed gene and 18S, GAPDH, and Actb for any given sample was defined as ΔC_T(X). The difference in ΔC_T(x) between two samples was defined as ΔΔC_T(X), which represents a relative difference in expression of the assayed gene. The fold change of the assayed gene relative to 18S, GAPDH, and Actb was defined as 2^−ΔΔCT.^{41 (link)} DataAssist software (Applied Biosystems) was used for statistical analysis and to confirm ΔC_T(X) calculation.

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Quantifying miRNA and Inflammatory Markers in Cells

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RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified using Nanodrop 2000 (Thermo). To measure miR-467 expression, 1–2.5 μg of total RNA was polyadenylated using NCode miRNA First-Strand cDNA Synthesis kit (Invitrogen) or miRNA 1st strand cDNA synthesis kit (Agilent). Real-time qPCR amplification was performed using SYBR GreenER™ qPCR SuperMix Universal (Thermo) or miRNA QPCR Master Mix (Agilent, Santa Clara, CA, USA). The miR-467 primer (GTA AGT GCC TAT GTA TATG) was purchased from IDT. To measure expression of inflammatory markers, 1–2 μg of total RNA was used to synthesize cDNA using the SuperScript First-Strand cDNA Synthesis System for RT-PCR (Invitrogen). Real-time qPCR was performed using TaqMan primers for Il6, Ccl2, Tnf, Cd68, Cd38, Egr2 (Thermo) and TaqMan Fast Advanced Master Mix (Thermo).

Gajeton J., Krukovets I., Muppala S., Verbovetskiy D., Zhang J, & Stenina-Adognravi O. (2021). Hyperglycemia-Induced miR-467 Drives Tumor Inflammation and Growth in Breast Cancer. Cancers, 13(6), 1346.

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Profiling miRNA and inflammatory markers

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RNA was isolated using Trizol reagent (Invitrogen) and quantified using Nanodrop 2000 (Thermo). To measure miR-467a expression, 1-2.5 µg of total RNA was polyadenylated using NCode miRNA First-Strand cDNA Synthesis kit (Invitrogen) or miRNA 1st strand cDNA synthesis kit (Agilent). Real-time qPCR amplification was performed using SYBR GreenER™ qPCR SuperMix Universal (Thermo) or miRNA QPCR Master Mix (Agilent). The miR-467a primer (GTA AGT GCC TAT GTA TATG) was purchased from IDT. To measure expression of inflammatory markers, 1-2 µg of total RNA was used to synthesize cDNA using the SuperScript First-Strand cDNA Synthesis System for RT-PCR (Invitrogen). Real-time qPCR was performed using TaqMan primers for Il6, Ccl2, Tnf, Cd68, Cd38, Egr2 (Thermo) and TaqMan Fast Advanced Master Mix (Thermo).

Gajeton J., Krukovets I., Muppala S., Verbovetskiy D., Zhang J., & Stenina‐Adognravi O. (2020). Hyperglycemia-Induced MIR-467 Drives Tumor Inflammation And Growth In Breast Cancer.

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Quantifying Pgβglu-1 Transcripts in Pinaceae

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Total RNA was extracted as in Chang et al. (1993) with modifications as in Pavy et. al. (2008) and stored at -80°C. The total RNA concentration was determined using a NanoDrop 1000 (Thermo Scientific) and assessed for quality with an Agilent 2100 Bioanalyzer and RNA 6000 Nano Kit LabChips (Agilent Technologies Inc.). Reverse transcriptase-qPCR with gene-specific primers was used to quantify Pgβglu-1 transcripts as described in Mageroy et al. (2015) . cDNA synthesis used 500 ng of total RNA and the Superscript First-Strand cDNA synthesis system for RT-PCR (Invitrogen). PCR reactions were performed using the QuantiFast SYBR Green PCR kit (Qiagen) as follows: 1x master mix, 300 nM of 50 and 30 primers and 5 µl of cDNA in a final volume of 15 µl. PCR reactions were carried out in a LightCycler 480 (Roche) as described in Boyle et al. (2009) . The linear regression of efficiency (LRE) method (Rutledge & Stewart 2008) was used to calculate the number of transcript molecules. Transcripts quantified with two different pairs of primers (N = 30, r = 0.97, P < 0.0001, Fig. S1) were highly correlated for P. abies as for P. glauca (Mageroy et al. 2015) . Similar results are expected in other species assayed for gene expression as sequences are highly similar across the Pinaceae (see results).

Parent G.J., Giguère I., Mageroy M., Bohlmann J, & MacKay J.J. (2018). Evolution of the biosynthesis of two hydroxyacetophenones in plants. Plant, cell & environment, 41(3).

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