mirVana™ miRNA Isolation Kit, with phenol

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercaptoethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation - To capture more amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step	Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA

Protocol tips
- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercaptoethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation - To capture more amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step
Downstream tips
Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA

Publication protocol

Total RNA was isolated using the following kits: Isolate II RNA mini kit (Bioline, London, UK), miRVana microRNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), Total RNA isolation kit (Norgen Biotek, Thorold, ON, Canada), miRNeasy mini kit (Qiagen, Hilden, Germany), and TRIzol Reagent (Thermo Fisher Scientific). Frozen tissue samples were weighed and processed on ice to prevent thawing. In preparation for RNA extraction 5–10 mg of tissue (weights differed by tissue but were consistent for all kits) was added to 700 μl of the appropriate lysis buffer. Samples were homogenised using a TissueLyser II (Qiagen) with 5 mm stainless steel beads for 3 × 1 min cycles at 30 Hz, with resting on ice in between. Homogenates were subjected to a single freeze/thaw cycle to aid cell lysis and then cleared by centrifugation at 10,000 x g for 5 min at 4 °C. RNA extractions were performed according to manufacturer’s instructions, with the exception of an additional chloroform extraction step for the TRIzol reagent protocol as illustrated in Fig. 2. RNA was eluted in 35 μl of RNase free water and stored at − 80 °C. Note that for all methods the total RNA extraction method was followed. miRNA enrichment was not performed for any method as efficient isolation of both miRNA and mRNA transcripts from the same sample is of interest.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for mirVana™ miRNA Isolation Kit, with phenol below.

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