The 10 basic tips & tricks presented in this slide-deck are based on Frequently Asked Questions raised by scientists, and their answers as supplied by the Ambion technical support teams at Life
The 10 basic tips & tricks presented in this slide-deck are based on Frequently Asked Questions raised by scientists, and their answers as supplied by the Ambion technical support teams at Life Technologies.
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.
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
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.
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.
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.
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.
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 .
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.
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.
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.