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

For all plasma and saliva samples we isolated 1 mL of biofluid. Samples were isolated using the mirVana miRNA Isolation Kit (ThermoFisher Scientific, AM1560) according to Burgos et al., 201341. Samples were DNase treated using TURBO DNA-free Kit (ThermoFisher Scientific, AM1907). Because of residual phenol/chloroform, samples were then cleaned and concentrated using Zymo RNA Clean and Concentrator (Zymo Research, R1016) using Protocol: Purification of small and large RNAs into separate fractions and combining the fractions at the end. All urine samples (15 mL) were isolated using Urine Total RNA Purification Maxi Kit, Slurry Format (Norgen Biotek Corp., Cat#29600). Samples were DNase treated on column using RNase-Free DNase Set (Qiagen, cat# 79254). Because there was no residual phenol/chloroform, samples were concentrated by Speed Vacuum.

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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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