miRNeasy Mini kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.

Protocol tips
- To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.

Publication protocol

The cells were lysed by the addition of 700 µL QIAzol Lysis Reagent. The phase separation was achieved by applying 2 mL of Phase Lock Gel Heavy (5PRIME Inc., Gaithersburg, MD, USA) to the lysates. RNA was then purified from the aqueous phase using the miRNeasy Mini Kit (Qiagen, Venlo, The Netherlands), according to the manufacturer's instructions. The RNA was eluted in 30 µL of RNase/DNase-free water and stored at −80°C until analysis. The A260/A280 ratio and RNA concentration were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The RNA quality was assessed by microfluidic capillary electrophoresis using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano Chip kit (Agilent Technologies, Santa Clara, CA, USA). An A260/A280 ratio in the range of 1.8 to 2.0 and an RIN>7 were considered acceptable. RNA samples were denatured for 2 min at 70°C prior to cDNA synthesis.

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Manufacturer protocol

Download the product protocol from Qiagen for miRNeasy Mini kit below.

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