MicroGEM Quick-Start Guide

RNA Extraction Using
RNAGEM

MicroGEM
Going to extremes for
effortless DNA extraction

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or email
info@microgembio.com

MicroGEM International PLC: Research Use Only. All products are subject
to a limited use license. See the product documentation on our website.

QSG_010_190531_RNAGEM

RNAGEM
When your kit arrives
All of the components in the RNAGEM kit are stable at room temperature and will arrive at your laboratory at ambient temperature.
However, once you have opened any tube or resuspended yourDNAse I in buffer, the components should be stored as follows:
10x Buffers
RNAGEM™ and DNAse I

4°C-20°C

Resuspending the DNAse I
DNAse I is delivered as a lyophilised powder. Before it is ready touse, the powder should be dissolved in 1x DNAse I Reaction Buffer(provided as a 10x solution). Different kit sizes contain tubes withdifferent amounts of enzyme (READ THE LABEL). Be sure to add
the correct amount of buffer (see the table below).
1.

Centrifuge the DNAse I tube for 1 minute at 10,000 x g. This
will settle the powder to the bottom of the tube.

2.

In a clean environment, open the tube and add:

DNAse Rxn Size
50 Rxn100 Rxn
200 Rxn
500 Rxn

10x DNAse Buffer
11 µl
25 µl
44 µl
105 µlRNAse-Free water99 µl
225 µl376 µl945 µl(MicroGEM supplies extra activity to cater for pipetting error)3.

Vortex and store at -20°C. The concentration of this solution
will be 1 Unit per µl.

Unit Definition: 1 Unit causes an increase in absorbance at 260nm of0.001 per minute per ml at 25°C when acting upon highly polymerised DNAat pH 5.0. 0.005 Kunitz unit digests 1 µg of lambda DNA in 10 minutes at37°C in 50 mM Tris, 1 mM Mg2+, pH 7.8 in a 50 µl reaction.

RNAGEM
Sample preparation and handling
RNAGEM is a kit for extracting total nucleic acids from mammaliantissue culture and is optimised for producing RNA. The methodlyses cells and digests proteins and ribonucleases. Extracted RNA
can be used for RT-PCR and RT-qPCR.
•All manipulations should be performed in an RNAse-freeenvironment or a PCR hood.

•Use only certified RNAse-free tubes and reagents.

RNAGEM gives linear yields for 10 to approx 105 cells and is ideal

for single-cell work. For low numbers of cells we recommend
reducing the extraction volume. The minimum volume possible
will depend on evaporation with the equipment you are using. The
recommended amounts of RNAGEM to use for different extraction
volumes are below. Use 1/10th volume of 10 X BLUE buffer.
Extraction Volume

Cell numbers

Volume of RNAGEM

50 - 100 µl
20 - 50 µl
5 - 20 µl
1 - 15 µl

50,000 - 500,000
5000 - 50,000
100 - 5000
1 - 500

1 µl
1 µl
0.5 µl0.2 µl

Sample handling will vary with different sample types. Anoutline of some suggested procedures is provided on theback page of this document. More information is available atwww.microgembio.com.
NOTE: RNAGEM is sensitive to EDTA and other chelating agents. Ifcells are presented in EDTA-containing solutions, they should becentrifuged at 200 x g and washed in 1X BLUE buffer before use.

Handling different culture types
Cells in suspension
1.
2.
3.

Centrifuge the suspension at 200 x g for 5 mins.
Remove all of the liquid.
Resuspend the pellet in RNAGEM extraction reagents.

Adherent cells
If the cells are in flasks, dislodge cells by preferred method (Trypsinor cell scraper) and centrifuge suspension at 200 x g for 5 mins.
Otherwise, the MicroGEM reagents can be added directly to theadhered layer.
1.
2.

Remove all of the liquid.
Add RNAGEM extraction reagents.

Cells stored in RNAlater™
1.
2.
3.

Centrifuge suspension at 3,000 x g for 5 mins.
Remove all of the liquid (a quick spin on a bench
centrifuge can help to gather the last few drops).
Resuspend the pellet in RNAGEM extraction reagents.

Cell pellets
Up to 5 x 105 cells can be extracted using the recommended
method. Linear extraction efficiency is best achieved within the rangeof <10 cells to approximately 105. Cell pellets can be used directly.
Alternatively, the pellet can be resuspended in 1X BLUE buffer and an
appropriate quantity added to the extraction.
FACS and LCM
Cells can be collected directly in the extraction reagent mastermix
or the reagents added directly to a capillary from LCM. If cells arecollected in a different buffer, it may be necessary to add 1/10th
volume of the MicroGEM buffer after collection. We recommend
using MicroGEM reagents within one hour of preparation. For longer
periods, reagents should be frozen.

Procedure(See notes on scaling for different volumes)
1. Add:Cell suspension or pellet
5 µl
10x BLUE Buffer
1 µl
RNAGEM
Water to a final volume of 50 µl
2. Vortex and incubate:
75°C for 5 min (< 50,000 cells) or 10 min (> 50,000 cells
4°C HOLD(A thermal cycler should be used for this step.)
DNAse treatment (IF REQUIRED)(Scale for different extraction volumes)
1.

To the extract add:				

5 µl of the 10x DNAse buffer
2 µl DNAse I

2. Vortex and incubate:						
3.

37°C for 5 minutes
75°C for 5 minutes
4°C HOLD

Add 1/10th volume of 10x TE Buffer (provided) and store at-20°C or below.

Technical tips and sample management•The method, enzyme formulation and buffer have been carefully
optimised for extracting intact RNA. Using the enzyme with othermethods or buffers is not recommended. If you need to modify themethod in any way, please email: support@microgembio.com.

•Absorbance 260/280 nm is an ineffective quantitation
method with RNAGEM-prepared nucleic acids. For accuratequantification we recommend RT-qPCR and normalisation togenomic DNA using a reference gene, or fluorometric based
assays.

•As with any method of RNA preparation, the best results areobtained when samples are handled on ice in an RNAse-freeenvironment and using certified RNAse-free tubes and reagents.

•For long-term storage, RNA should be stored at -80°C.

•Alternatively, RNA in TE buffer can be precipitated using NH4OAc/
ethanol (0.1 volumes of 5 M NH4OAc, and 2.5 volumes of 100%ethanol) and stored at -20°C or below.

More information is available on our website at www.microgembio.com