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

Total RNA was extracted using miRNeasy kit (Qiagen) with some modifications. The collected pellets were washed with SET buffer [50 mM NaCl, 5 mM EDTA and 30 mM Tris-HCl (pH 7.0)]. After centrifugation at 16000×g for 3 min pellets were resuspended in 0.1 ml Tris-HCl (pH 6.5) containing 50 mg/ml lysozyme (Sigma), 25 U of mutanolysin (Sigma), 40 U of SUPERase (Ambion), 0.2 mg of proteinase K (Ambion) and incubated at 37°C for 30 min at 350 rpm. QIAzol (Qiagen) was added, mixed gently and incubated for 3 min at room temperature. An additional incubation at room temperature was done after adding 0.2 volume chloroform followed by centrifugation at 16000×g at 4°C for 15 min. The aqueous phase, containing RNA, was transferred to a new collection tube and 1.5 volumes of 100% ethanol was added and mixed thoroughly. The probes containing RNA were transferred into columns supplied with the miRNeasy Kit (Qiagen) and treated according to the manual including an on-column DNase digestion (RNase-Free DNase, Qiagen). RNA was eluted by RNase-free water and stored at −80°C until needed. The quantity of the isolated total RNA was determined by absorbance at 260 nm and 280 nm, and the quality was assessed using Nano-chips for Agilent's 2100 Bioanalyzer. For detection and estimation of the small RNA fraction within the isolated total RNA, a small RNA-chip (Agilent) was used, which visualizes RNAs with sizes ranging from 20 to 150 nucleotides.

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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