Total RNA was extracted using TRIzol (Life Technologies) and RNeasy Plus mini kit (Qiagen). Total RNA was reverse transcribed using oligo(dT) primers with the Superscript II Synthesis Kit (Life Technologies). qPCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. Expression levels were calculated using the ΔΔCt method and normalized to GAPDH. Real-time qPCR was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) and analyzed with the StepOne Software v2.2.2. Primers used in qPCR assays are listed in Table S2 .
Superscript 2 synthesis kit
Manufactured by Thermo Fisher Scientific
The Superscript II Synthesis Kit is a laboratory equipment product designed for reverse transcription, which is the process of converting RNA into complementary DNA (cDNA). The kit contains the necessary reagents and enzymes to perform this RNA-to-cDNA conversion.
Automatically generated - may contain errors
Lab products found in correlation
Fast sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Rneasy plus isolation kit, by Qiagen (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Phusion polymerase, by New England Biolabs (1 mentions)
5 protocols using superscript 2 synthesis kit
1
Quantitative Gene Expression Analysis
2
Quantifying Cardiac Gene Expression in Human Engineered Cardiac Tissues
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To determine gene expression of the tissues, total RNA was extracted from the hECTs using Trizol (Life Technologies) and the RNeasy plus isolation kit (Qiagen). RNA was reverse transcribed using oligo-dT primers with the Superscript II Synthesis Kit (Life Technologies). qPCR was completed with Fast SYBR Green Mastermix (Applied Biosystems) according to the manufacturer’s instructions. The primers used were cardiac troponin (cTnT), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a). Gene expression was normalized against the housekeeping gene GAPDH using the ΔΔCt method. The expression level of cardiac genes is reported relative to expression of cardiac-specific cTnT. A StepOne Plus Real-Time PCR System (Applied Biosystems) was used to perform the qPCR and analyzed with StepOne Software v2.2.2.
3
Quantitative Analysis of Cardiomyocyte Transcripts
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Total RNA was extracted using Trizol (Invitrogen) and RNeasy plus mini kit (Qiagen) from hiPSCs, EBs, and sorted redhigh and redlow cardiomyocytes. Total RNA was reverse transcribed using oligo-dT primers with the Superscript II Synthesis Kit (Invitrogen). qPCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Expression levels were calculated using the ΔΔCT method and normalized to GAPDH. Real time qPCR was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) and analyzed with the StepOne Software v2.2.2. Primers and conditions used in qPCR assays are listed in Table S2
4
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was isolated using Trizol reagent (Sigma, Aldrich) according to manufacturer’s protocol. Isolated RNA was then treated with DNase using Ambion DNA-free kit and 0.5–2 µg of RNA was used for cDNA synthesis using the Invitrogen’s SuperScript II synthesis kit. Quantitative RT-PCR was performed on an Applied Biosystems 7100 as we have previously described [45] (link). Sequences for the primers are presented in Table 3 ; all are selected using Primer Express software (Aplied Biosystems). Samples were normalized to the housekeeping gene Gapdh. Levels of transcript were extrapolated from a standard curve constructed with five known concentrations ranging from 10 copies/µl to 100,000 copies/µl. Data were expressed as number of copies/µg RNA.
5
Nup Protein Deletion Construct Generation
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The total RNA was isolated from rat spleen using the Trizol reagent (Invitrogen), and cDNA was prepared with the Superscript-II synthesis kit (Invitrogen) following the manufacturer’s instructions. The gene-specific primers (Supplemental Table S6) for Nup88517-742, Nup62322-525, and Nup214693-976 were used to amplify various deletion constructs using Phusion polymerase (NEB). The α-helix of Nup62322-525 and the α-helix as well as β-propeller regions of Nup8859-498 and Nup2141-407 were cloned into the respective vectors (refer to Supplemental Table S6). In all cases, affinity tags were removed by thrombin digestion. All positive clones were confirmed by gene sequencing.
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