TaqMan® MicroRNA Reverse Transcription Kit

siRNA / miRNA gene silencing Mouse - Glomerular mesangial cells HIPK2 Polymer / Lipid delivery

Experiment

siRNA / miRNA gene silencing Mouse - Glomerular mesangial cells HIPK2 Polymer / Lipid delivery

Product

TaqMan® MicroRNA Reverse Transcription Kit from Thermo Fisher Scientific

Manufacturer

Thermo Fisher Scientific

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Protocol tips

Upstream tips	Protocol tips	Downstream tips
	cDNA was synthesized using TaqMan® microRNA Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA), combined with Stem-loop RT Primers (Life Technologies) and High Capacity RNA-to-cDNA Master Mix (Life Technologies), according to the manufacturer's guidelines. For miRNA, 3 µl (10 ng) total RNA was mixed with 3 µl specific primers, 100 mmol l−1 dNTPs, 50 µl MultiScribe™ Reverse Transcriptase, 10× RT Buffer and 20 µl RNase inhibitor, and made up to a final volume of 4.5 µl with H2O. The conditions were 16°C for 2 min, 42°C for 1 min, 50°C for 1 s and 85°C for 5 min. For mRNA, 10 µl total RNA was mixed with 4 µl Master Mix, 2 µl specific primers and made up to a final volume of 20 µl with H2O. The conditions were 25°C for 5 min, 42°C for 30 min and 85°C for 5 min.

Protocol tips
cDNA was synthesized using TaqMan® microRNA Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA), combined with Stem-loop RT Primers (Life Technologies) and High Capacity RNA-to-cDNA Master Mix (Life Technologies), according to the manufacturer's guidelines. For miRNA, 3 µl (10 ng) total RNA was mixed with 3 µl specific primers, 100 mmol l−1 dNTPs, 50 µl MultiScribe™ Reverse Transcriptase, 10× RT Buffer and 20 µl RNase inhibitor, and made up to a final volume of 4.5 µl with H2O. The conditions were 16°C for 2 min, 42°C for 1 min, 50°C for 1 s and 85°C for 5 min. For mRNA, 10 µl total RNA was mixed with 4 µl Master Mix, 2 µl specific primers and made up to a final volume of 20 µl with H2O. The conditions were 25°C for 5 min, 42°C for 30 min and 85°C for 5 min.

Protocol tips

cDNA was synthesized using TaqMan® microRNA Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA), combined with Stem-loop RT Primers (Life Technologies) and High Capacity RNA-to-cDNA Master Mix (Life Technologies), according to the manufacturer's guidelines. For miRNA, 3 µl (10 ng) total RNA was mixed with 3 µl specific primers, 100 mmol l−1 dNTPs, 50 µl MultiScribe™ Reverse Transcriptase, 10× RT Buffer and 20 µl RNase inhibitor, and made up to a final volume of 4.5 µl with H2O. The conditions were 16°C for 2 min, 42°C for 1 min, 50°C for 1 s and 85°C for 5 min. For mRNA, 10 µl total RNA was mixed with 4 µl Master Mix, 2 µl specific primers and made up to a final volume of 20 µl with H2O. The conditions were 25°C for 5 min, 42°C for 30 min and 85°C for 5 min.

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Papers

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Manufacturer protocol

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