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-mercapto ethanol 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-mercapto ethanol 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 and miRNA isolation was performed using RNeasy kit (Qiagen, USA) and mirVana miRNA Isolation Kit (Ambion, USA) respectively as per manufacturer recommendations. 1 μg of total RNA and miRNA was used for cDNA synthesis in 20 μl reaction using Verso Reverse Transcriptase (Thermo Scientific, USA) and miScript Reverse Transcription Kit (Qiagen, Valencia, CA, USA) respectively as per manufacturer’s protocol. 2 μl of cDNA was used to analyze the mRNA/miR-31 expression using gene specific primers in Real Time Thermocycler (Stratagene, USA, MX3005P). Single peak of dissociation curve from the amplified produced was considered as specific amplification.

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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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