Total RNA was isolated with peqGOLD Total RNA Kit (PeqLab) and reverse transcribed using the Superscript II RT First Strand Kit (Invitrogen). Real time PCR was conducted in the ABI 7500 Fast Real-Time PCR cycler (Applied Biosystems) using a SYBR Green I Low Rox Mastermix (Eurogentec GmbH) and the respective primers (Supplementary Table S2 ). Following a 10 min denaturing step at 95°C 40 cycles with 15 seconds 95°C and 1 min 60°C were applied. Primer sequences and PCR efficiencies are given in Supplementary Table S2 .
Superscript 2 rt first strand kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Superscript II RT First Strand Kit is a laboratory product designed for the reverse transcription of RNA into complementary DNA (cDNA). It provides the necessary reagents and enzymes to efficiently convert RNA into single-stranded cDNA, which can then be used for various downstream applications, such as PCR amplification and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Peqgold total rna kit, by Avantor (2 mentions)
Abi 7500 fast real time pcr cycler, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Accuprime taq polymerase system, by Thermo Fisher Scientific (1 mentions)
Takyon low rox sybr mastermix, by Eurogentec (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
4 protocols using superscript 2 rt first strand kit
1
Quantitative Real-Time PCR for Gene Expression
2
RNA Isolation and RT-PCR Analysis
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Total RNA was isolated using the RNeasy Mini Kit (Qiagen GmBH, Hilden, Germany). From each sample, 2 μg of RNA was converted into cDNA by oligo(dT)18-primed reverse transcription using SuperScript II RT First-Strand kit (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. The cDNA was subject to semiquantitative PCR analysis using Accuprime Taq polymerase system (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s recommendations. Flt3 primers were used as previously described in Ref. (8 (link)).
3
Gene Expression Analysis of Stem Cell Markers
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Total cellular RNA was isolated by using the peqGOLD total RNA kit (VWR; Darmstadt, Germany) with a subsequent DNaseI digestion step according to the manufacturer’s instructions. For cDNA synthesis, the Superscript II RT First Strand Kit (Invitrogen GmbH, Karlsruhe) was used. PCR primer sequences used to detect ATOH1, GLI1, KRT8, 14, 17, 18, 20, RPLP0, SOX2, and SOX9 are given in Supplementary Methods . Thermal profile for the PCR using the Takyon Low Rox Sybr MasterMix (Eurogentec; Cologne, Germany) contained an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of two-step PCR including 15 sec at 95 °C and 60 sec at 60 °C. Quantification was performed in three independent experiments.
4
Quantitative Analysis of HSP70 Isoforms
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Relative expression levels of the HSP70 isoforms were determined by SybrGreen real time PCR applying the comparative ΔΔ−CT method. Total cellular RNA was isolated using the RNAeasy kit (Qiagen, Hilden, Germany) with a subsequent DNaseI digestion step according to the manufacturer's instructions followed by cDNA synthesis with the Superscript II RT First Strand Kit (Invitrogen GmbH, Karlsruhe).
Real time PCR was conducted in the ABI 7500 Fast Real-Time PCR cycler (Applied Biosystems Inc., Foster City, CA, USA). The standard PCR reactions (20 μl) contained 1 μl cDNA and 10 μl 2× SybrGreen I Low Rox Mastermix (Eurogentec GmbH, Cologne, Germany) for detection. The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of three-step PCR including 15 sec at 95 °C, 60 sec at 60 °C and 30 sec at 95 °C. The CT levels of the investigated HSP70 isoforms were normalized to GAPDH (Δ-CT level). Δ-CT was calculated as CT HSP isoform, sample - CT GAPDH, sample.
Primer pairs for the different HSP70 isoforms were selected from NCI qPrimerDepot (Table 1 ) (http://primerdepot.nci.nih.gov/ ).
Real time PCR was conducted in the ABI 7500 Fast Real-Time PCR cycler (Applied Biosystems Inc., Foster City, CA, USA). The standard PCR reactions (20 μl) contained 1 μl cDNA and 10 μl 2× SybrGreen I Low Rox Mastermix (Eurogentec GmbH, Cologne, Germany) for detection. The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of three-step PCR including 15 sec at 95 °C, 60 sec at 60 °C and 30 sec at 95 °C. The CT levels of the investigated HSP70 isoforms were normalized to GAPDH (Δ-CT level). Δ-CT was calculated as CT HSP isoform, sample - CT GAPDH, sample.
Primer pairs for the different HSP70 isoforms were selected from NCI qPrimerDepot (
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