P ROTOCOLRNA extraction from tissue using Bioruptor® (Standard/Plus) and RNA extraction kit
Introduction
Isolation of intact RNA is essential for many techniques used in gene expression analysis. Efficient
disruption and homogenization of animal tissues are required to ensure high yield of RNA. Disruption
releases RNA, while homogenization reduces sample viscosity to facilitate RNA purification.
Diagenode’s Bioruptor® Sonicator uses state-of-the-art ultrasound technology to efficiently disrupt
and homogenize tissues in one step. Diagenode’s RNA extraction reagent (included in the RNAextraction kit) is used as sonication medium and maintains the integrity of RNA while disrupting
cells and dissolving cell components.
Bioruptor® together with RNA extraction reagent offer unique benefits for tissue disruption andhomogenization:•

Fast protocol•

Non-contact minimizes contamination•

Efficient and reproducible•

Isothermal process•

Multiplexing capability of up to 6 samples in parallel (depends on Bioruptor® model)

General remarks before starting•

Minimize the time of tissue collection to prevent RNA degradation by RNases and fromchanges in RNA expression triggered by sample manipulation. RNA quality correlates totissue-specific response to physiological stress both prior to and following tissue death.

•Dissected tissues can be snap-frozen in liquid nitrogen and stored at -80°C until RNA
extraction. Alternatively, RNAlater solution can be used to protect RNA in unfrozen sample.

•

This protocol has been validated for 20-50 mg of tissue. Do not use more tissue per sample
as it might result in low quality RNA. For larger quantity, cut the tissue and proceed todisruption in separate tubes.

•When working with RNA extraction reagent, work in fume hood and use gloves and eye
protection.

•When working with RNA, care must be taken to maintain an RNase-free environment startingwith RNA purification and continuing through analysis. Wear gloves at all times to prevent
RNase contamination from the surface of the skin or from dusty laboratory equipment.
Change gloves frequently and keep tubes closed whenever possible. Keep isolated RNA on
ice when aliquots are pipetted for downstream applications.

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Required materials and reagents•Bioruptor® Standard or Plus (Diagenode, Cat. No. UCD-200, UCD-300) for tissue disruption
and homogenization•

RNA extraction kit (Diagenode, Cat. No. AL-001-0050)•Bioruptor® Water Cooler (Diagenode, Cat. No. BioAcc-Cool)•Single Cycle Valve for Bioruptor® Plus (Diagenode, Cat. No. VB-100-0001)•Tube holder pack for extraction kits (Diagenode, Cat No. O-ring-15)•Liquid nitrogen or RNAlater solution (Ambion, Cat #7020) for tissue collection•Isopropanol (Molecular Biology grade)•Chloroform (Molecular Biology grade)•70 % ethanol (Molecular Biology grade)•RNase-free water•

2 ml RNase-free tubes•

Nanodrop and Agilent Bioanalyzer for quality assessment (optional)

Protocol
Tissue disruption and homogenization
1. Pre-cool Bioruptor to 4°C using the Bioruptor® Water Cooler (Cat. No. BioAcc-Cool) or crushed
ice.
2. Prepare sonication tubes: add 1 ml of cold RNA extraction reagent to the pre-filled RNA extraction
tubes.
We highly recommend to use these tubes without reflecting bars.

3. Keep the tubes on ice.
4. Add 20-50 mg of snap-frozen tissue per tube containing RNA extraction reagent. Alternatively,
RNAlater-treated samples can be used.
If needed, cut the frozen tissue in a Petri dish placed on dry ice. Minimize the time required to do this, anddo not allow sample to thaw before immersing into RNA extraction reagent.

5. Adjust the sample volume with RNA extraction reagent to final volume 2 ml and vortex vigorously.
6. Insert the aluminium rings to ensure an optimal position of the tube in tube holder duringsonication (see picture). To guarantee homogeneity of sonication, the tube
holders should always be completely filled with tubes.
7. Sonicate samples in Bioruptor using the following settings:• Power: H position (High)• Sonication cycle: 30 seconds ON, 30 seconds OFF• Temperature: 4°C• Total sonication time: 1-3 cycles
8. Stop Bioruptor® after each cycle, vortex samples and visually check thesample for disruption.
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Please note that optimization might be required depending on sample format (fresh or frozen tissue),tissue type and tissue amount. The shortest sonication time should be chosen to prevent RNA degradation.
Incomplete disruption may occur with fibrous tissues like muscles. Do not sonicate longer than 3 cycles toprevent low quality RNA.
Samples with high amount of blood, iron or hemoglobin may change the blue color of RNA extraction
reagent.

9. Vortex tubes vigorously after sonication and incubate for 5 min at room temperature.
RNA isolation
10. Centrifuge samples at 3.000 rpm for 5 min at room temperature and transfer the supernatant toa new 2 ml RNase-free tube.
This step permits the complete dissociation of nucleoproteins complex

11. Add 0.4 ml of chloroform, vortex and centrifuge at 12.000 g for 10 min at 4°C.
Chloroform mixed with isoamyl alcohol should not be used.
Thorough mixing is important for subsequent phase separation

12. Transfer the colorless upper phase to a new 2 ml tube. Take care not to aspirate the DNAcontaining white interface and organic blue phase.
If contamination of genomic DNA is expected, extract again by adding an equal volume of chloroform to theaqueous phase transferred to the new tube.

13. Add 0.8 ml of isopropanol, mix and centrifuge at 12.000 g for 10 min at 4°C. A gel-like pellet
forms on the side and bottom of the tube.
14. Remove the supernatant and keep the pellet.
15. Add 1 ml of 70% ethanol, vortex samples and centrifuge for 10 min at 12.000 g
16. Remove the supernatant and air-dry the pellet for 5-10 min at room temperature. Do not overdry the pellet.
17. Add RNase-free water (100-300 μl depending on expected RNA yield), resuspend carefully bypipetting. The solution can be incubated at 55-60 C° for 10 min if the pellet is hard to dissolve.
18. Take an aliquot for quantitation and quality analysis. Store RNA -80°C.
RNA quantitation and quality assessment
19. Quantify RNA using a Nanodrop and analyze ratio OD 260/280 and OD 260/230 to ensure thepurity of RNA.
Ratio OD260/280 1.8-2.0 is considered good. A low ratio might indicate protein contamination. A ratio
greater than 2.1 might indicate RNA degradation.
Ratio OD260/230 greater than 1.8 is considered good. A low value might indicate organic contamination.

20. Asess the integrity of RNA using the Agilent 2100 Bioananlyzer (or BioRad Experion system). .
RIN values threshold depends on the desired downstream experiments and should be correlated with thespecific assay to be run (RT-PCR or microarray, for example).
The table below shows suggested applications for RNA within different RIN ranges:
RIN ValueRNA quality

Suggested application

1-4degraded to low

PCR assays with short regions of amplification

4.1-6.9

moderate to regular

qRT-PCR applications

7.0-10.0

excellent to outstanding

Highly demanding gene array assays

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Example of tissue disruption

Figure 1.
An example of mouse brain disruption using RNA extraction tubes and RNA extraction reagent.
Left picture shows samples before sonication. Right picture shows disrupted sample.

Examples of total RNA profiles obtained from animal tissues
snap-frozen

RNAlater-stored

Figure 2.
Total RNA efficiently extracted from snap-frozen (lanes 1-5) and RNAlater-treated (lanes 6-11) mouse brain
samples.
Tissue was disrupted with the Bioruptor® Standard (UCD-200). Total RNA was extracted as directed in theprotocol and analyzed on BioAnalyzer (Agilent).

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Figure 3.

RIN 8.0
Brain

Efficient extraction ofpure RNA with high RIN.
Total RNA profiles frommousebrain(upperpanel), liver (middle panel)
andskeletalmuscle(bottom panel). Tissues
weredisrupted
withBioruptor Plus (UCD300) as described in theprotocol and analyzed
on BioAnalyzer (Agilent).
Note that small RNAs
are present in all profiles
indicating that the RNA islargely intact.

RIN 7.6
LiverRIN 7.6
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P ROTOCOLRQI 5.7- RNA extraction beads

RQI 7.8+ RNA extraction beads

Figure 4.
Pre-filled RNA extraction tubes improve tissue disruption and RNA quality.
Experion (BioRad) traces of total RNA obtained from mouse liver using Bioruptor and the RNA extraction
kit without (upper panel) or with RNA extraction beads (bottom panel). Note that only 2 cycles are requiredfor complete tissue disruption using RNA extraction beads vs 15 cycles without RNA extraction beads. RNA
extracted from a sample disrupted in the presence of RNA extraction beads shows significantly higher RQI.

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Troubleshooting guide
ProblemLow yields
Expected yield of RNA per mgof tissue:

Possible cause

Suggested solution

RNA is not solubilized
completelyDo not allow pellet to drycompletely. Do not lyophilize orvacuum dry sample.

Sample manipulated too muchbefore freezing or RNAlater
stabilization

Process tissue
after dissection

Liver 4-10 µg
Skeletal muscle 0.3-1 µg
Brain 2-5 µg
HeLa cells 6-15 µg per
1X10^6 cells
Degraded RNA

Improper storage of RNA
Frozen tissue thawed inabsence of RNA extraction
reagent
Sample sonicated too much

Low RIN value due to high DNA/protein contamination isbaseline
possible
RIN 5.6

immediately

Store RNA at -80°C avoid thawfreeze cycles
Add frozen tissue immediately
to RNA extraction reagent
Avoid long sonication.1-2 cycles
are enough. Do not sonicate
longer even if some particulatematter remains.
Be sure not to take any of theinterphase (contains DNA) withthe aqueous phase
Do not use more than 50 mg for
2 ml of RNA extraction reagent.
Removematerialaddition

any
beforeparticulate
chloroform

Treat with RNase-free DNase I(see additional protocol)
Low ratio OD 260/280

Protein contamination

Too much tissue used. Do notuse more than 50 mg for 2 mlof RNA extraction reagent.
Be sure not to carry any organic
phase with the RNA sample(step 9 in the protocol)

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Low ratio OD 260/230

Organic contamination(chloroform, polysaccharidesetc)

Too much tissue used. Do notuse more than 50 mg for 2 ml
of RNA extraction reagent.
Be sure not to carry any organic
phase with the RNA sample(step 9 in the protocol)

RNA contains some DNA

Part of the interphase was Be sure not to take any of theremoved with the aqueous interphase (contains DNA) withphase
the aqueous phase
Too much tissue used for 2 ml Do not use more than 50 mg forof RNA extraction reagent
2 ml of RNA extraction reagent.
Insoluble material was not Remove
removed before extraction
materialaddition

any
beforeparticulate
chloroform

For use in PCR and qRTPCR, treatment with DNase I(RNase-free) is recommended

Additional protocol for DNase I treatment
Combine up to 10 µg of RNA, 1 µl of RNase-free DNase (1 U/µl), 5 µl of 10X DNase buffer, 1µl of RNasin (optional) and RNase-free water to final volume of 50 µl•Incubate sample for 15-30 min at room temperature•

Add EDTA to final concentration 2 mM

•Extract RNA samples with 100 µl of RNA extraction reagent and 20 µl of chloroform. Use 80 µlof isopropanol to precipitate RNA•

Wash the pellet once with 70% ethanol, air-dry and resuspend in RNase-free water

PRO-RNA-EXTRACT_21_05_12•© 2012 Diagenode SA. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in
retrieval systems, or translated into any language or computer language. The trademarks mentioned herein are the propertyof Diagenode or their respective owners. Bioruptor is a registered trademark of Diagenode SA. Diagenode RNA extraction
reagent is powered by Isogen. Bioanalyzer is a trademark of Agilent Technologies, Inc. Experion is a trademark of Bio-Rad.

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