mirVana™ miRNA Isolation Kit, with phenol

RNA isolation / purification Tissue - Human Salivary glands

Experiment
RNA isolation / purification Tissue - Human Salivary glands
Product
mirVana™ miRNA Isolation Kit, with phenol from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

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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Discussion

Discussion

1 year ago

Author: Paul G. Macon United States

RNA isolation from tissue

How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?

Discussion

2 years ago

Author: Aaron Stege Netherlands

Problem in phase separation after using serum/plasma kit

I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?

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Papers

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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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