miRNA Isolation Kit

For research use only

Sample: up to 100 mg of tissue, up to 1 x 106 cultured cells
RXNS: 50
Format: phenol/chloroform/spin column purification
Time : within 30 minutes
ISO 9001:2008 QMS
Elution volume: 50-100 µl
Storage: dry at room temperature (15-25ºC) for up to 9 months

Introduction
The miRNA Isolation Kit provides a quick and easy spin column system for purifying and enriching micro RNAs
(miRNAs) and other small cellular RNAs from a wide variety of tissue and cells. Since miRNAs are vital for
regulating gene expression, this kit is optimized for isolation of small RNA molecules while removing larger RNAs
and minimizing genomic DNA contamination for improved sensitive downstream applications.

Quality Control
The quality of the miRNA Isolation Kit is tested on a lot-to-lot basis according to Geneaid's ISO-certified quality
management system. Purified miRNA is resolved in 50 µl of Release Buffer and a 1/10 volume aliquot (5 µl) is
analyzed by electrophoresis on a 2% agarose gel.

Kit Contents Order Information

1
Add absolute ethanol (see the bottle label for volume) to the Wash Buffer prior to initial use

Caution
Buffers contain harmful irritants. During operation, always wear a lab coat, disposable gloves, protective goggles
and (anti-fog) procedure mask.

Steps to prevent RNase contamination

Disposable and nondisposable plasticware and automatic pipettes should be sterile and used only for RNA
procedures.

References
(1) Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615

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miRNA Isolation Kit Protocol
• Additional Requirements: Trypsin, phosphate-buffered saline (PBS), ddH20 saturated phenol, chloroform, absolute ethanol,
microcentrifuge tubes, pipette tips, (optional) RNase-free water

Tissue
• Remove frozen tissue samples from storage or excise fresh tissue samples.
• Transfer up to 100 mg of fresh or frozen tissue to a 1.5 ml microcentrifuge tube then proceed to Step 1 Lysis.
NOTE: Ensure frozen tissue does not thaw prior to adding Lysis Buffer.
Adherent Cultured Animal Cells
• Remove the culture medium and wash cells in PBS.
Sample
• Aspirate PBS then add 0.10-0.25% Trypsin in PBS.
Preparation
• Once cells have detached, add the medium and transfer to a 15 ml centrifuge tube.
• Proceed with Suspension Cultured Animal Cells.
Suspension Cultured Animal Cells
6
• Transfer cells (up to 1 x 10 ) to a 1.5 ml microcentrifuge tube.
• Harvest by centrifugation for 5 minutes at 300 x g.
• Carefully remove the supernatant completely by aspiration then proceed to Step 1 Lysis.
Tissue: Add 200 µl of Lysis Buffer then use a Micropestle to grind the tissue until it is dissolved completely.
Step 1 Cultured cell pellet: Add 200 µl of Lysis Buffer then vortex vigorously until the pellet is dissolved completely.
Lysis • Incubate at room temperature for 10 minutes.
At this time, pre-heat the required Release Buffer (50 µl/sample) to 65ºC (for Step 5 Elution).
• Add 20 µl of Mi Buffer.
• Add 180 µl of ddH2O saturated phenol and 40 µl of chloroform.
Step 2
• Vortex vigorously for 2 minutes then centrifuge at 14-16,000 x g for 3 minutes.
RNA
• Transfer the upper phase to a clean 1.5 ml microcentrifuge tube.
Precipitation
• Add a 35% volume of absolute ethanol to the upper phase and mix well by shaking vigorously.
If the upper phase volume is 200 µl, 108 µl of absolute ethanol should be added, e.g. 108/(200+108)=0.35.
• Place a RNA Column in a 2 ml Collection Tube and transfer the ethanol-added mixture to the RNA Column.
• Incubate for 1 minute at room temperature then centrifuge at 14-16,000 x g for 30 seconds.
• Transfer the filtrate to a new 1.5 ml microcentrifuge tube.
Step 3
• Add a 70% volume of absolute ethanol to the filtrate and mix well by shaking vigorously.
RNA
If the filtrate volume is 290 µl, 676 µl of absolute ethanol should be added, e.g. 676/(290+676)=0.70.
Binding
• Place a new RNA Column in a 2 ml Collection Tube then transfer the mixture to the RNA Column.
• Incubate for 1 minute at room temperature.
• Centrifuge at 14-16,000 x g for 30 seconds to allow the miRNA to bind to the RNA Column membrane.
• Add 200 µl of Wash Buffer (make sure ethanol was added) to the RNA Column.
Step 4 • Incubate for 1 minute at room temperature.
Wash • Centrifuge at 14-16,000 x g for 1 minute to completely remove the liquid residue.
• Place the RNA Column in a clean 1.5 ml microcentrifuge tube.
• Add 50 µl of Release Buffer (pre-heated to 65ºC) into the CENTER of the RNA Column.
• Incubate for 3 minutes at room temperature.
Step 5
• Centrifuge at 14-16,000 x g for 3 minutes to recover the miRNA.
Elution
The purified miRNA can be further concentrated using a standard ethanol precipitation procedure then
redissolved in a small volume of RNase-free water.
Use a 1/5 volume to run on a polyacrylamide gel to check the quality. The majority of RNA visible on the gel
should be <100 nt in size, with the major bands corresponding to tRNAs. The 5S and 5.8S rRNA species may
QC Analysis
also be visible. These tRNA and small rRNA bands should be clear and distinct. miRNA (21-22 nt) are typically
not visible on the gel image.

Troubleshooting
Problem Possible Reasons/Solution
• Insufficient disruption and/or homogenization
Clogged
• Too much starting material
Column
• Centrifugation temperature was too low (should be 20ºC to 25ºC)
• Insufficient disruption and homogenization
Low RNA
• Too much starting material
Yield
• RNA still bound to the RB Column membrane
• Ethanol carryover
• Harvested sample not immediately stabilized
RNA
• Inappropriate handling of starting material
Degradation
• RNase contamination

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