1. Immediately inactivate endogenous, intracellular RNases.
   Endogenous RNases must be inactivated immediately upon tissue harvesting and cell death to prevent RNA degradation.
   
   There are 3 effective methods to accomplish this:
   
   
   1. Homogenize samples immediately after harvesting in a chaotropic-based cell lysis solution (e.g. containing guanidinium).
      
      
   2. Flash freeze samples in liquid nitrogen. In order to inactivate RNase by flash freezing, it is important that tissue pieces be small enough to freeze almost immediately upon immersion in liquid nitrogen.
      
      
   3. Place samples in RNAlater™ Tissue Collection: RNA Stabilization Solution, an aqueous, nontoxic collection reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue and cell samples (see #2 below). It is essential that tissue samples be in thin pieces (0.5 cm) so that the RNAlater can quickly permeate the tissue before RNases destroy the RNA.
   
   
   
2. Use proper cell or tissue storage conditions.
   When samples have been flash frozen they must be stored at -80°C and never allowed to thaw. Even brief thawing prior to homogenization in a guanidinium-based lysis solution can result in RNA degradation and loss. Flash frozen tissues should be ground or pulverized at cryogenic temperatures prior to homogenization in a lysis solution.
   
   RNAlater offers much greater flexibility for storage. Cells or tissues can be harvested into RNAlater and stored at room temperature for up to 1 week, at 4°C for up to 1 month, or at -20°C indefinitely. For more information on RNAlater, read www.ambion.com/techlib/resources/RNAlater.
   
   
3. Thoroughly homogenize samples.
   Thorough homogenization of cells or tissues is an essential step in RNA isolation that prevents both RNA loss and RNA degradation. The method of homogenization should be tailored to the cell or tissue type. Whereas most cultured cells can be homogenized by simply vortexing in a cell lysis solution, animal tissues, plant tissues, yeast, and bacteria often require more rigorous methods of disruption. Bacterial cell walls for example, may require enzymatic digestion to achieve thorough cell lysis and maximum recovery of RNA. For detailed information about which disruption method is ideal for many sample types read www.ambion.com/techlib/tb/tb_183.html.
   
   
4. Pretreat homogenate before RNA isolation.
   Additional treatments are needed for some samples after homogenization and before RNA isolation. Lysates made from tissues high in fat, like brain and adipose tissue, should be extracted with chloroform to remove lipids and increase RNA yields. Many plant tissues are high in polyphenolics and polysaccharides that can decrease RNA quality and yield. Pretreatment of the lysate with the Plant RNA Isolation Aid removes these troublesome compounds
   
   
5. Choose the best RNA isolation method.
   With all of the different RNA isolation methods available it can be difficult to decide which one to use. The easiest and safest methods available are column-based methods like RNAqueous™ or RNAqueous-4PCR. Due to ease of handling, these procedures are ideal for working with multiple samples. When working with difficult tissues, for example ones that are high in nucleases (pancreas) or fat (brain and adipose tissue), a more rigorous, phenol-based RNA isolation method like or ToTALLY RNA™ is recommended. For additional information read www.ambion.com/techlib/tn/83/8311.html.
   
   
6. DNase treatment.
   When the RNA will be used for RT-PCR, we recommend treating it with DNase to remove residual contaminating DNA. DNase treatment is also a good idea when isolating RNA from tissues that are high in DNA, like spleen. Ambion's RNAqueous-4PCR Kit includes a DNase treatment as part of the protocol, and is supplied with the necessary reagents. The DNA-free™ DNase treatment & Removal Reagents can be used to remove contaminating DNA from RNA purified by any method. Both products offer high quality DNase I, an optimized reaction buffer, and a quick and easy way to remove the DNase after the treatment without using organic solvents or risking a heat treatment.
   
   
7. Reduce exposure to environmental RNases.
   To isolate intact, high quality RNA, it is essential that RNases are not introduced into RNA preparations once they are no longer protected by strong protein denaturants such as a chaotropic lysis solution or phenol. Since RNases are found almost everywhere, 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 like RNaseZap™ or RNaseZap Wipes. RNase-free tips, tubes, and solutions should always be used and gloves should be changed frequently.
   
   
8. Proper precipitation.
   The purified RNA may need to be concentrated by precipitation for downstream applications. An ammonium acetate (NH₄OAc) precipitation (0.1 volumes of 5 M NH₄OAc, and 2-2.5 volumes 100% ethanol, at -20¨C for >25 min) 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 could introduce nucleic acid contamination into samples, potentially skewing RT-PCR results. After precipitation, avoid complete drying of the RNA pellet because it can make RNA difficult to resuspend.
   
   
9. Resuspension.
   The final step in many RNA isolation procedures is to suspend the purified RNA pellet. The 3 ideal qualities of a resuspension solution are that it be RNase-free, have a low pH (pH 6-7), and incorporate a chelating agent to protect against RNA degradation by introduced RNases. (THE RNA Storage Solution meets all of these criteria.) To aid solubilization, the RNA pellet should be incubated in resuspension solution at 65¨C for 5 min with intermittent gentle vortexing.
   
   
10. Storage.
    For short-term storage, resuspended RNA should be stored at -20°C; for long-term storage, it 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 NH₄OAc/ethanol precipitation mixture at -80°C. We recommend aliquotting RNA solutions into several tubes. This will both prevent damage to the RNA from successive freeze-thaw events, and help to prevent accidental RNase contamination.
    

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