Ambion's Top Ten:
Tips for RNA Isolation

Act fast.
Successful RNA isolation requires fast processing and careful handling of the tissue or cells prior to isolation. Endogenous RNases are released from cellular compartments immediately after harvesting tissue. It is essential to inactivate these RNases as soon as possible to prevent RNA degradation; any one of the following methods is effective for RNase inactivation:

• Homogenize samples immediately after harvesting in a chaotropic-based cell lysis solution (e.g. a guanidinium solution).

• Place samples in Ambion’s RNAlater®, an aqueous, nontoxic reagent that stabilizes and protects RNA in intact, unfrozen tissue and cell samples. Tissue samples should be trimmed into thin pieces (0.5 cm) so that the RNAlater can quickly permeate the tissue.

• Flash-freeze samples in liquid nitrogen. To prevent RNA degradation, it is important that the tissue be cut into small enough (1 cm) pieces to allow rapid, thorough freezing of the entire tissue.

Choose the RNA isolation method to suit your needs.
With all of the different RNA isolation methods available, it can be difficult to decide which one to use. (See Which Kit to Choose for an overview of Ambion's RNA Isolation Kits.) The easiest and safest methods available are filter-based methods such as Ambion’s RNAqueous® Kit. Due to the ease of handling, filter-based procedures are ideal for working with multiple samples. When working with tissues high in nucleases (e.g. liver, pancreas) or high in fat, a phenol-based method (to eliminate protein), such as the RiboPure™ Kit, or ToTALLY RNA™ is recommended. To extract RNA from paraffin embedded tissues a protease digestion step is necessary.

Determine the quality of RNA needed.
Northern analysis, cDNA library construction, and cDNA labeling for microarray analysis all require RNA of extremely high integrity. RT-PCR and ribonuclease protection assays both involve analysis of smaller regions of RNA and, therefore, are more tolerant of partially degraded RNA. Samples to be used in RT-PCR or cDNA sythesis/labeling have higher requirements for purity (i.e. no inhibitors of enzyme reactions).

Homogenize thoroughly.
Thorough sample homogenization maximizes RNA recovery and prevents RNA degradation. The method of homogenization should be tailored to the cell or tissue type under study: cultured cells can be homogenized by vortexing in a cell lysis solution, whereas animal tissues, plant tissues, yeast, and bacteria usually require more rigorous methods of disruption. For more information on selecting a homogenization method, see the Technical Bulletin Cell Disruption: Getting the RNA Out.

Check RNA integrity.
Regardless of the downstream application, it is a good idea to check RNA integrity before gene expression analysis. Intact total RNA run on a denaturing gel will have sharp ribosomal RNA bands (e.g., 28S and 18S rRNA in human samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S rRNA) is a good indication that the RNA is intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will have a lower 28S:18S rRNA ratio. Highly degraded RNA will appear as a very low molecular weight smear.

Although usually adequate, visual assessment of the 28S:18S rRNA ratio on an agarose gel can be somewhat problematic because the appearance of rRNA bands is affected by electrophoresis conditions, amount of RNA loaded, and saturation of ethidium bromide fluorescence. The best analytical tool currently available for total RNA analysis is the Agilent 2100 bioanalyzer, which uses a combination of microfluidics, capillary electrophoresis, and fluorescence to evaluate both RNA concentration and integrity. Another advantage is that it requires very small volumes, allowing the user to assay RNA quality in limiting samples.

Remove genomic DNA.
When the RNA will be used for RT-PCR or array analysis, we recommend treating it with DNase to remove residual DNA. DNase treatment is also a good idea when isolating RNA from tissues, such as spleen, that are high in DNA. Ambion's TURBO DNA-free™ provides reagents to remove DNA, then to remove the DNase and divalent cations after digestion without the need for phenol extraction or alcohol precipitation.

Reduce exposure to exogenous RNases.
To isolate intact, high quality RNA, it is essential that RNases are not introduced into purified RNA preparations. Because of the ubiquitous presence of RNases, it is essential that any item that could contact the purified RNA is RNase-free. All surfaces, including pipettors, benchtops, glassware, and gel equipment, should be decontaminated with a surface decontamination solution such as RNaseZap® or RNaseZap Wipes. RNase-free tips, tubes, and solutions should always be used and gloves should be changed frequently. The use of disposable plasticware in place of reused glassware can reduce the risk of carryover contamination.

Maximize recovery of RNA concentrated by precipitation.
The purified RNA may have to be concentrated by precipitation for downstream applications. Ethanol precipitation in the presence of ammonium acetate gives good recovery of RNA. For quantitative recovery of low concentrations of RNA (ng/ml), an inert coprecipitant (e.g., glycogen, yeast RNA, or linear acrylamide) should be used. Linear acrylamide and DNase-treated glycogen are the coprecipitants of choice when the RNA will be used in RT-PCR because they do not contain contaminating DNA. Yeast RNA and untreated glycogen will introduce nucleic acid contamination into samples, potentially skewing RT-PCR results. After precipitation, avoid complete drying of the RNA pellet because it can make the RNA difficult to resuspend. Ambion offers a collection of RNase-free coprecipitates.

Thoroughly resuspend the RNA pellet after precipitation.
The final step in many RNA isolation procedures is to suspend the purified RNA pellet. A resuspension solution should be RNase-free, have a low pH (pH 6–7), and incorporate a chelating agent to protect against divalent cation catalyzed RNA degradation. (THE RNA Storage Solution™ meets all of these criteria.) To improve solubilization, the RNA pellet can be incubated in the resuspension solution at 65°C for 5 min, then vortexed.

Store RNA properly.
For short-term storage, RNA can be stored at –20°C; for long-term storage, it should be stored at –80°C. Although RNA can be stored in water or buffer, it is most stable in an NH₄OAc/ethanol mixture at –80°C. We recommend aliquotting RNA solutions into several tubes to prevent damage to the RNA from successive freeze-thaws and to reduce the risk of introducing RNases into the tube.

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