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

Gene expression profiling of BxPC3 and BxPC3-R cells was performed using GeneChip miRNA 3.0 Array (Affymetrix, Santa Clara, CA). Cellular RNA enriched for small RNAs (<200 nt) was isolated using the mirVana miRNA isolation kit according to the manufacturer's instructions (ThermoFisher Scientific). The concentration and quality of miRNA were determined using the Small RNA Assay in the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). In all, 400 ng of cellular RNA enriched for small RNAs was labeled using the FlashTag Biotin HSR Labeling Kit, hybridized for 18 h at 48 °C (rotation 60 r.p.m.) on the GeneChip miRNA 3.0 Array, washed in the Affymetrix Fluidics Station 450 and scanned using the Affymetrix GeneChip Scanner 3000 7G (all from Affymetrix) as previously described.12

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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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