RNA isolation was performed following the manufacturer’s instructions using RNeasy Lipid Tissue Mini Kit (QIAGEN, Germany, N = 6). Subsequent cDNA synthesis was performed using the Molecular Biology RevertAid Strand cDNA Kit (Thermo Fisher Scientific, Germany) with anchored oligo (dT)18 primers. Quantstudio Applied Biosystems (Thermo Fisher Scientific, Germany) and SYBR green PCR master mix (+Rox, Roche, Germany) were used for qPCR analysis. The most stable reference genes were determined with geNorm version 3.420 (link) and gene expression levels were normalized with hypoxanthine phosphoribosyltransferase (Hprt) and cyclophilin D (Cyclo). The expression of uncoupling protein 1 (Ucp1), β3-adrenergic receptor (Adrb3) and deiodinase 2 (Dio2) were measured and results were calculated using the ∆∆CT method, and standard curves were recorded to correct for PCR efficiency. Primer sequences are available on request.
Molecular biology revertaid strand cdna kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Molecular Biology RevertAid Strand cDNA Kit is a reagent kit designed for the synthesis of first-strand cDNA from RNA templates. The kit includes all the necessary components for the reverse transcription reaction, including a RevertAid Reverse Transcriptase enzyme, reaction buffer, and random hexamer primers.
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2 protocols using molecular biology revertaid strand cdna kit
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RNA Isolation and qPCR Analysis
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RNA Isolation and qPCR Analysis
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RNA isolation was performed on snap-frozen tissue using RNeasy Kits (QIAGEN, Germany). Subsequent cDNA synthesis was carried out using the Molecular Biology RevertAid Strand cDNA Kit (Thermo Fisher Scientific, Germany). Quantstudio Applied Biosystems (Thermo Fisher Scientific, Germany) and SYBR Green PCR Master Mix (Roche, Germany) were used for qPCR analysis. Gene expression levels were normalized with one or two housekeeping genes (Hprt/Gapdh for pituitary, Hprt/Cyclo for liver, Hprt/Cyclo for hypothalamus and Hprt for gWAT) and PCR efficiency was corrected by the calculation of standard curves.
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