Briefly, total RNA, including microRNA, was isolated from tissue using the Invitrogen mirVana miRNA isolation kit (A1561, ThermoFisher) following the manufacturer's instructions. Total RNA was analyzed using a NanoDrop 1000 to ensure appropriate 260/280 and 260/230 ratios. The total RNA concentration was measured using a Qubit 2.0 fluorometer (ThermoFisher). Reverse transcription with gDNA digestion was performed using 0.5–1.0 μg total RNA with the QuantiTect RT Kit (205,313, Qiagen) following the manufacturer's instructions. An RT Negative control was used to confirm the elimination of gDNA. All qPCR assays were run with a LightCycler96 (Roche) with FastStart Essential DNA Green Master (Roche) following the manufacturer's instructions for cycling parameters. Fold expression of mRNA transcripts was determined by the comparative CT method (Livak and Schmittgen, 2001 (link)).
Invitrogen mirvana mirna isolation kit
Manufactured by Thermo Fisher Scientific
The Invitrogen mirVana miRNA isolation kit is a product designed for the extraction and purification of microRNA (miRNA) from various biological samples. The kit utilizes a proprietary method to selectively isolate small RNA molecules, including miRNA, from total RNA.
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2 protocols using invitrogen mirvana mirna isolation kit
1
Quantitative Real-Time PCR Analysis
2
Quantitative Real-Time PCR Analysis
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Briefly, total RNA, including microRNA, was isolated from tissue using the Invitrogen mirVana miRNA isolation kit (A1561, ThermoFisher) following the manufacturer's instructions. Total RNA was analyzed using a NanoDrop 1000 to ensure appropriate 260/280 and 260/230 ratios. The total RNA concentration was measured using a Qubit 2.0 fluorometer (ThermoFisher). Reverse transcription with gDNA digestion was performed using 0.5–1.0 μg total RNA with the QuantiTect RT Kit (205,313, Qiagen) following the manufacturer's instructions. An RT Negative control was used to confirm the elimination of gDNA. All qPCR assays were run with a LightCycler96 (Roche) with FastStart Essential DNA Green Master (Roche) following the manufacturer's instructions for cycling parameters. Fold expression of mRNA transcripts was determined by the comparative CT method (Livak and Schmittgen, 2001 (link)).
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