Steps for Obtaining High-Quality RNA

Our ongoing research into optimizing RNA preparation and analysis has identified several points in the process that can commonly be improved and are often overlooked:

• Treatment and handling of samples prior to RNA isolation
• Choice of technologies used to prepare the RNA
• Storage of the prepared RNA sample

Most traditional RNA isolation procedures take place in the presence of RNase inhibitory agents (typically strong denaturants like guanidine salts, sodium dodecylsulfate (SDS), or phenol-based compounds that are designed to lower the risk of RNA degradation in a sample). However, it is typically prior to and after the isolation when RNA integrity is at highest risk.

Finding the most appropriate method of cell or tissue disruption for your specific starting material is important for maximizing the yield and quality of your RNA preparation. During sample disruption for RNA isolation, it is crucial that the lytic agent or denaturant be in contact with the cellular contents at the moment that the cells are disrupted. This can be problematic when tissues or cells are hard (e.g., bone, roots), when they contain capsules or walls (e.g., yeast, gram-positive bacteria, spores), when workflows prevent processing immediately after collection (e.g., transport from a site of collection to another location for processing), or when samples are numerous (making rapid processing difficult). A common solution to these problems is to freeze the tissue/cells in liquid nitrogen or on dry ice. The frozen samples are often preprocessed to select a desired mass or to partially pulverize the sample before exposure to denaturant. While this freezing and preprocessing allows the researcher more control over the purification conditions, our experience and feedback from customers confirm that this is a complex, time-consuming, and laborious process.

RNAlater® reagent and RNAlater®-ICE Sample Storage: RNA Stabilization Solutions provide more flexibility and time to allow the researcher to postpone RNA isolation for days, weeks, or even months after tissue collection, without sacrificing the integrity of the RNA. Dissected tissue, body fluids, or collected cells are simply introduced into the RNAlater® solution at room temperature, or into RNAlater®-ICE solution, if frozen. The solution permeates the cells and stabilizes the RNA. The samples are then stored at 4°C using RNAlater® reagent, or at –20°C when using RNAlater®-ICE RNA Stabilization Solution. Samples can be shipped on wet ice or even at room temperature if shipped overnight. Figure 1 shows the integrity of RNA isolated from tissues stored in RNAlater® reagent at 4°C, room temperature, and even at 37°C for increasing lengths of time. Samples stored at 4°C generate intact RNA, even after storage for a month.

Speed is critical in the purification of bacterial RNA due to the short half-life of bacterial mRNA and the need to rapidly "freeze" the mRNA expression profile. Some bacterial isolation protocols call for the pretreatment of bacteria with lytic enzymes (which are usually used in conjunction with a one-step isolation reagent such as TRI Reagent). While this pretreatment does assist lysis, it delays isolation and may lead to altered expression profiles.

The following methods are better alternatives for effectively freezing gene expression profiles:

• Immediate cell lysis and RNA purification
• Rapid freezing in liquid nitrogen (a freeze-thaw treatment may help with lysis of some bacteria)
• Resuspension of cells in Ambion's RNAlater

Easily lysed Gram-negative bacteria may be pipetted directly into a boiling lysis buffer of choice (without even removing the culture medium), and RNA can be immediately extracted with TRI Reagent. Most other bacteria will need to be pelleted by brief centrifugation prior to the above treatments.

To purify RNA from a bacterial cell pellet (including frozen pellets and those stored in RNAlater), add boiling lysis buffer to the pellet and vortex rapidly, then immediately extract the lysate with TRI Reagent or hot acid phenol:chloroform. Harsh mechanical devices (e.g. bead mills) may be required to disrupt some bacterial species. Once lysed, extract the preparation with hot acid phenol:chloroform or TRI Reagent. Alternatively, it may be possible to disrupt bacteria directly in TRI Reagent or acid phenol:chloroform using a bead mill. For more information see "Purify Bacterial RNA".

Eukaryotic mRNA
Poly(A) RNA (mRNA) makes up between 1-5% of total cellular RNA and is most frequently used for 1) detection and quantitation of extremely rare mRNAs, 2) synthesis of probes for array analysis, and 3) the construction of random-primed cDNA libraries, where the use of total RNA would generate rRNA templates that would significantly dilute out cDNAs of interest. Removal of ribosomal and transfer RNA results in up to a 30-fold enrichment of a specific message. Figure 6 demonstrates the enrichment of a specific message seen after selecting for poly(A) RNA.



Ambion's Poly(A)Purist™ Kits make isolation of mRNA easy by providing a rapid method for isolating the highest possible purity mRNA from total RNA, with the highest possible yield. The Poly(A)Purist and MicroPoly(A)Purist Kits include premeasured aliquots of oligo(dT) cellulose. The Poly(A)Purist MAG Kit utilizes oligo(dT) magnetic bead-based purification. These kits use an optimized hybridization protocol so that mRNA is efficiently bound but without co-isolation of rRNA. These procedures usually require only a single round of oligo(dT) selection to yield mRNA for even the most stringent applications.

Prokaryotic mRNA
For decades mRNA has been isolated from eukaryotic sources using oligo(dT) selection. Bacteria, however, lack the relatively stable poly(A) tails found on eukaryotic messages. Until very recently, isolating mRNA from bacteria has been virtually impossible.

The MICROBExpress™ Bacterial mRNA Isolation Kit employs a novel technology to remove >95% of the 16S and 23S rRNA from total RNA of E. coli and other bacterial species The kit is suitable for rapid mRNA purification from a broad spectrum of Gram-positive and Gram-negative bacteria. mRNA isolated with MICROBExpress is a superior template for synthesizing labeled cDNA for array analysis (Figure 7) and is ideal for quantitative RT-PCR, Northern blotting, and cDNA library construction.



In the first step of the MICROBExpress procedure, bacterial total RNA is mixed with an optimized set of capture oligonucleotides that bind to the bacterial 16S and 23S rRNAs. Next, the rRNA is removed from the solution using derivatized magnetic microbeads. The mRNA remains in the supernatant and is recovered by ethanol precipitation. The entire procedure takes less than 2 hours.

The last step in every RNA isolation protocol, whether for total or mRNA preparation, is to resuspend the purified RNA pellet. After painstakingly preparing an RNA sample, it is crucial that RNA be suspended and stored in a safe, RNase-free solution. Ambion now has several RNA Storage Solutions for this purpose:

• THE RNA Storage Solution (1 mM sodium citrate, pH 6.4 ± 0.2)
• >0.1 mM EDTA (in DEPC-treated ultrapure water)
• TE Buffer (10mM Tris-HCl, 1 mM EDTA, pH 7.0)
• RNAsecure™ Resuspension Solution

Our technical service staff has received numerous requests for pre-made 0.1 mM EDTA and TE Buffers; these solutions are often specified in common RNA isolation and analysis protocols. These storage solutions are ideal for researchers who already use them but would like the convenience and security of having them premade and certified RNase-free.

We are also introducing THE RNA Storage Solution, a buffer which delivers greater RNA stability than 0.1 mM EDTA or TE. THE RNA Storage Solution has two features which minimize base hydrolysis of RNA: a low pH, and sodium citrate, which acts both as pH buffer and a chelating agent (divalent cations catalyze base hydrolysis of RNA). THE RNA Storage Solution is compatible with all of the common RNA applications such as reverse transcription, in vitro translation, Northern analysis, and nuclease protection assays.

The RNAsecure Reagent is a unique nonenzymatic reagent for the irreversible inactivation of RNases in enzymatic reactions. RNAsecureª Resuspension Solution contains the same active ingredients as the RNAsecure Reagent, but is supplied at a working concentration for direct resuspension of RNA pellets. To inactivate RNases, the RNA pellet is resuspended in the RNAsecure Resuspension Solution and heated to 60°C for 10 minutes. A unique feature of RNAsecure is that reheating after the initial treatment will reactivate the RNase-destroying agent to eliminate any new contaminants.

Ambion is continuously inventing ways to make RNA analysis easier. Our goal is to provide unique products to solve the problems researchers frequently face when working with RNA. Ambion's RNAlater Solution, RNA Isolation Kits, and RNA Storage Solutions are designed to work together to take you all the way from tissue to your RNA analysis application.