RNA Isolation: The Basics

Obtaining high quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments, including Northern analysis, nuclease protection assays, RT-PCR, RNA mapping, in vitro translation and cDNA library construction. To be successful, however, the RNA isolation procedure should include some important steps both before and after the actual RNA purification. The following article discusses various RNA isolation procedures and ways of increasing RNA yields.

Tissue or Cell Sample Collection and Disruption
Our ongoing research into optimizing RNA analysis has identified two points in the RNA isolation process that can be improved; treatment and handling of tissue or cells prior to RNA isolation and storage of the isolated RNA. Since most of the actual RNA isolation procedure takes place in a strong denaturant (e.g. GITC, LiCl, SDS, phenol) that renders RNases inactive, it is typically prior to, and after the isolation, when RNA integrity is at 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. See the article "Cell Disruption - Getting the RNA Out," which describes various disruption methods and suggests which method to use for specific tissues / cell types, for more information on this subject.

During tissue disruption for RNA isolation, it is crucial that the denaturant be in contact with the cellular contents at the moment that the cells are disrupted. This can be problematic when tissues/cells are hard (e.g. bone, roots), when they contain capsules or walls (e.g. yeast, Gram-positive bacteria) 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 samples must then be ground with a mortar and pestle into a fine powder, which is added to the denaturant. While this freezing and grinding process allows the researcher to postpone RNA isolation, it is a time consuming and laborious process.

Ambion now offers a completely new type of product, a tissue storage solution that provides more flexibility and time. RNAlater® Tissue Storage:RNA Stabilization Solution allows the researcher to postpone RNA isolation for days, weeks, or even months after tissue collection without sacrificing the integrity of the RNA. Dissected tissue or collected cells are simply dropped into the RNAlater solution at room temperature. The solution permeates the cells, stabilizing the RNA. The samples are then stored at 4°C. 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 at 4°C, room temperature and even at 37°C. Samples stored at 4°C generate intact RNA even after storage for a month.

Figure 1. Quality of RNA Isolated from Tissue Stored in RNAlater®. RNA was purified from the tissues using TRIzol® (Invitrogen). Five µg of each RNA sample was run on a denaturing agarose gel. The top panel shows ethidium bromide-staining of the gel. The bottom panel shows a Northern blot of the same gel hybridized with 10⁶ cpm/ml of a high specific activity RNA probe to p53 and 10⁴ cpm/ml of a low specific activity RNA probe to GAPDH, using Ambion's NorthernMax® Kit reagents and protocol.

Use of RNAlater for tissue storage is compatible with most RNA isolation procedures. Tissues stored in RNAlater are simply removed and processed by homogenization via a dounce, Polytron® (Brinkman), or other mechanical apparatus in the lysis buffer specified by your RNA isolation procedure. See the article "Cell Disruption - Getting the RNA Out" for a discussion of sample disruption. Figure 2 shows the RNA isolated from tissue stored in RNAlater Solution using several methods, and Figure 3 demonstrates that mRNA signal intensities in RPAs are not affected by storage in RNAlater.

Options for RNA Isolation
Ambion's family of RNA isolation kits provide flexibility for sample size and type, and include kits for the isolation of total or poly(A) RNA. The accompanying table — "Which RNA Isolation Kit to Choose?" — summarizes the advantages of each kit to help you determine the optimal RNA Isolation Kit for your particular application. This table also gives information on typical yield per reaction. For additional information on approximately how much total or poly(A) RNA can be recovered from a given amount of tissue or cells, as well as rough expression levels for rare to moderately abundant transcripts, see "Tissue/Cells to RNA Conversions."

Total RNA
Eukaryotic Total RNA
Ambion provides several options for isolation of total eukaryotic RNA that are compatible with a variety of cells and tissues, including yeast, plants and animals.

TRI Reagent® is a single, homogenous solution for the isolation of total RNA. This phenol-based reagent contains a unique combination of denaturants and RNase inhibitors and is used in a convenient, single-step disruption/separation procedure. The tissue or cell sample is homogenized or disrupted in the TRI Reagent, chloroform is mixed with the lysate, and the mixture is separated into three phases by centrifugation. The RNA is then precipitated from the aqueous phase with isopropanol. The entire procedure can be completed in no more than one hour to produce high yields of intact RNA for use in Northerns, nuclease protection assays, RT-PCR and in vitro translation. TRI Reagent is especially effective for purifying RNA from microorganisms.

Ambion's RNAqueous® Technology is a rapid, filter-based RNA isolation system that does not require the use of phenol, chloroform or other toxic organic chemicals. The entire procedure can be completed in 20 to 30 minutes, depending on the time required for tissue disruption (see an example of typical results in Figure 4). RNAqueous Technology-based Kits are available in both small and large scale formats. The RNAqueous Kit is designed for sample sizes of 10 to 75 mg of tissue or 10⁶ to 10⁷ cells, whereas the RNAqueous-Midi Kit is designed for tissue samples of up to 0.5 g or 10⁹ cells. The RNAqueous-96 Kit utilizes a 96-well plate format for high-throughput RNA isolation from 100 to 2 x 10⁶ cells or 0.1 to 1.5 mg of tissue. The RNAqueous-4PCR Kit provides RNA free of genomic DNA contamination from samples as small as 1 mg or 100 cells. Ambion's Plant RNA Isolation Aid is recommended for use with the RNAqueous Kit for purification of total RNA from plant tissues.

Alternatively, total RNA may be isolated using Ambion's ToTALLY RNA™ Kit. This procedure is similar to the widely used guanidinium thiocyanate/acid phenol:chloroform method but has been modified to include two formulations of phenol:chloroform and an optional LiCl precipitation step. The modifications help to reduce or eliminate DNA, carbohydrate, heme and other contaminants that can otherwise be difficult to separate from the RNA. Although more time consuming than other RNA isolation procedures — it can take up to 85 minutes from tissue/cell disruption to RNA — reactions can be scaled up or down to accommodate large or small samples.

The Paraffin Block RNA Isolation Kit allows easy isolation of RNA from formalin-fixed, paraffin-embedded tissue sections for use in RT-PCR. The fast, 4-hour protocol yields RT-PCR competent RNA even for rare messages (Figure 5). Paraffin-embedded tissue blocks as old as 16 years have yielded amplifiable RNA.


Figure 5. Differentially Expressed Messages Amplified from RNA Isolated using the Paraffin Block RNA Isolation Kit. Total RNA was isolated from two 20 µm mouse heart tissue sections (6-month old block; tissue fixed in 10% neutral buffered formalin for 6 hrs) as per protocol. Twelve distinct genes were amplified by RT-PCR. Fragments were electrophoresed on a 2% agarose gel.

Prokaryotic Total RNA
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:

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".

mRNA
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.


Figure 6. Enrichment of mRNA after Oligo(dT) Selection. The indicated amounts of total RNA from mouse liver, the fraction of RNA that did not bind to oligo dT, and the poly(A)RNA were hybridized with a 300 base antisense RNA transcript for mouse GAPDH. Total yeast RNA was added so that each sample contained 5 µg of RNA. The samples were hybridized and digested with RNase under standard conditions for the RPA II™ Kit, leaving only mRNA:probe hybrids. The resulting 250 base protected mRNA:probe duplex was precipitated and run on a 5% polyacrylamide/8 M urea gel and analyzed by autoradiography. Lane 1, puc19/Sau 3A markers; lane 2, probe alone control; lane 3, probe + yeast RNA + RNase; lanes 4-8, dilutions of total RNA; lanes 9-13, dilutions of the unbound fraction of total RNA incubated with oligo dT; lanes 14-18, dilutions of poly(A) RNA. (Note: The concentration of poly(A) RNA used was 1/10th the amount examined with total 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.

Figure 7. Array Analysis Using E. coli mRNA Purified with the MICROBExpress™ Kit. Replicate Sigma Genosys Panorama™ E. coli Gene Arrays were hybridized with ³³P-labeled cDNA synthesized from equal mass amounts (1.5 µg) of either E. coli mRNA purifed with MICROBExpress or E. coli total RNA. Random primers were used to prepare labeled cDNA. Arrays were hybridized with equal cpm of labeled cDNA, following the array manufacturer's instructions. There is a dramatic increase in the hybridization signal, but no change in the expression profile when labeled cDNA is prepared from bacterial mRNA instead of from total RNA.

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:

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. If you have suggestions for additional products that would be useful in your RNA research, please contact us.