The document outlines essential tips for working with RNA, addressing concerns such as inactivating RNases, proper sample storage, homogenization techniques, and RNA isolation methods. It emphasizes the importance of quality assessment, contamination elimination, and environmental precautions to protect RNA integrity. Additionally, it highlights the need for careful experimental design and integration of high-quality reagents into workflows for reliable results.

1. Basic tips & tricks for
working with RNA
Based on Frequently Asked Questions raised by scientists

2. tip #1 Inactivate endogenous RNases
tip #2 Proper sample storage conditions
tip #3 Thorough sample homogenization
tip #4 Optimal RNA isolation
tip #5 Eliminate contaminating gDNA
tip #6 Exposure to environmental RNases
tip #7 Assess RNA quality
tip #8 Storage of RNA
tip #9 Integrate into workflow
tip #10 Experimental design

3. tip #1
Inactivate endogenous RNases

4. tip #1
Inactivate endogenous RNases
- Should be done immediately upon sample harvesting:
Homogenize samples in a chaotrope-based cell lysis solution such
as TRIzol®. This will effectively inhibit RNase activity while disrupting cells
and cell components during sample homogenization.
Flash-freeze samples in liquid nitrogen / dry-ice alcohol bath.
Tissue pieces must be small enough to freeze immediately upon immersion.
Place samples in RNAlater® Tissue Collection & RNA Stabilization
Solution. This aqueous, nontoxic reagent stabilizes and protects cellular
RNA in intact, unfrozen tissue and cell samples. Tissue samples must be in
thin pieces (≤0.5 cm thick) to allow the solution to quickly permeate before
RNases destroy the RNA.

5. tip #2
Proper sample storage conditions

6. tip #2
Proper sample storage conditions
-Flash-frozen sample must be stored at –80°C, even brief thawing prior to
homogenization/lysis can result in RNA degradation and loss.
-Flash-frozen tissue should be ground/pulverized at cryogenic
temperatures prior to homogenization in a lysis solution .
-RNAlater® Solution offers great flexibility for storage. Cells or tissues can
be harvested and stored at:
room temperature for up to 1 week
4°C for up to 1 month
long term at –20°C

7. tip #3
Thorough sample homogenization

8. tip #3
Thorough sample homogenization
-Homogenization methods should be tailored to the specific cell/tissue type:
Most cultured cells can be homogenized by vortexing in lysis
solution.
Animal/plant tissues, yeast, and bacteria require more rigorous
disruption, such as a rotor-stator homogenizer or a French Press.
Lysis of single-celled organisms is often aided by the addition of
hydrolytic enzymes specific for cell wall components to achieve maximum
recovery of RNA.

9. tip #4
Optimal RNA isolation

10. tip #4
Optimal RNA isolation
-Depends on sample type, application, throughput and personal preferences:
Solid-phase extraction to a thick filter matrix (column), such as the
PureLink™ RNA Mini Kit.
For tissue rich in e.g. nucleases (pancreas) or fat (brain/adipose tissue),
or applications demanding protein removal, use the TRIzol® Plus RNA
Purification System. Chaotropic salts and a strong denaturing solvent
strips off proteins. Can be used with a second purification.
Magnetic separation technology for high throughput requirements,
manual or automated methods:
• MagMAX™ nucleic acid isolation kits for genomic and viral nucleic acid from diverse
samples (cells, tissue, blood and cell-free media).
• Dynabeads® Oligo(dT)25 is available alone, and as part of different kits for isolation of mRNA
directly from virtually any crude lysate.

11. tip #5
Eliminate contaminating gDNA

12. tip #5
Eliminate contaminating gDNA
-RNA for use in PCR, real-time PCR or array hybridizations should be treated
with DNase, as the DNA can interfere with the RNA signal.
-RNA from DNA-rich tissue (e.g. spleen) should also be treated with DNase .
-PureLink™ DNase Set (lyophilized RNase-free DNase) removes DNA from
RNA purified using PureLink™ RNA kits. Optimized for on-column digestion of
DNA using PureLink™ protocols.
-Ambion®’s Turbo DNase-free kit™ removes DNA in just 5 minutes.

13. tip #6
Exposure to environmental RNases

14. tip #6
Exposure to environmental RNases
-Ensure no RNases are introduced into RNA preparations once they are no
longer protected by the strong protein denaturants used for extraction.
-RNases are found almost everywhere!
-Any item that may come into contact with the RNA must be RNase-free:
Decontaminate all surfaces (e.g. pipettes, bench-tops, glassware, and
gel equipment) with a surface decontamination solution such as
RNaseZap® or RNaseZap® Wipes.
Change gloves frequently.
Always use RNase-free tips, tubes, and solutions.

15. tip #7
Assess RNA quality

16. tip #7
Assess RNA quality
-UV absorbance readings will measure anything that absorbs at 260 nm (incl.
DNA, protein, degraded nucleic acids, and free nucleotides).
-Qubit® 2.0 Fluorometer uses fluorescent dyes to quantitate, and can
distinguish between RNA and the different contaminants . This generates
more accurate and precise results across a lower concentration range.
-Electrophoretic analysis of total RNA to assess the rRNAs. The relative area
of the large subunit (23-28S, depending on species) to the small subunit (16-
18S) is a good indication, as the larger rRNA will degrade faster.
-E-Gel® EX gels for RNA sample integrity analysis.
-For total RNA samples analyzed on an Agilent 2100 Bioanalyzer, the
software can be used to determine the RIN value (RNA Integrity Number) on
a scale of 1-10, where 10 is ideal .
-For fractionated RNA samples (e.g. poly(A)+ RNA), the type of sample must
be considered, and often functional tests must be used (e.g. Northern blots or
qRT-PCR). qRT-PCR will often work fine with partially degraded samples .

17. tip #8
Storage of RNA

18. tip #8
Storage of RNA
-For short-term storage, RNA should be stored at –20°C.
-For long-term storage, RNA should be stored at –80°C .
-Although RNA resuspended in water or buffer can be stored at –80°C, RNA
is most stable in an Ammonium Acetate /ethanol precipitation mixture kept at
–80°C .
-Aliquot your RNA into several tubes to prevent RNA damage from successive
freeze-thaw events and to prevent accidental RNase contamination.

19. tip #9
Integrate into workflow

20. tip #9
Integrate into workflow
-Regardless of the quantity and quality of RNA obtained, the downstream
results can be critically impacted by the specific reagents used.
-Life Technologies offers the best-in-class, validated reverse transcriptase
enzymes and reagents for real-time PCR.
-By using SuperScript® VILO™ cDNA Synthesis Kit with high quality isolated
RNA, your real-time PCR data will be the best it can be.

21. tip #10
Experimental design

22. tip #10
Experimental design
-Always consider the overall experimental design to ensure validity.
-Run ≥3 biological replicates with technical replicates of the analytical
process. Can be reduced if the biological specimens are in an ordered series
(e.g. dosing levels or time points), where statistical vigor can be obtained by
the modeling of the response .
-Carefully matched controls provide a baseline that may change between
experiments, providing a more robust output, and helping to differentiate noise
from the interesting data.

23. Learn more at
lifetechnologies.com/ambion