Donald C. Rio

Purification of RNA using TRIzol (TRI reagent).

TRIzol solubilization and extraction is a relatively recently developed general method for deproteinizing RNA. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when separation of cytoplasmic RNA from nuclear RNA is impractical. TRIzol (or TRI Reagent) is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously solubilizes biological material and denatures protein. After solubilization, the addition of chloroform causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase. Therefore, RNA, DNA, and protein can be purified from a single sample (hence, the name TRIzol). TRIzol extraction is also an effective method for isolating small RNAs, such as microRNAs, piwi-associated RNAs, or endogeneous, small interfering RNAs. However, TRIzol is expensive and RNA pellets can be difficult to resuspend. Thus, the use of TRIzol is not recommend when regular phenol extraction is practical.

Northern Blots for Small RNAs and MicroRNAs

This protocol describes the detection of small RNAs (~10-200 nucleotides) by blot hybridization. The RNA samples, denatured in formamide, are separated by denaturing polyacrylamide gel electrophoresis. Because high-percentage polyacrylamide gels are required to separate RNAs in this size range, it is necessary to perform electrophoretic transfer to positively charged nylon membranes. After transfer, the immobilized RNAs are subjected to hybridization with a (32)P-radiolabeled DNA or RNA probe and detected by phosphorimaging or autoradiography. This procedure is commonly used to detect small, U-rich spliceosomal small nuclear RNAs (snRNAs) and miRNAs. It should be possible also to detect most miRNAs using high-percentage (e.g., 15%) urea-polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis of RNA

Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. This technique is generally applicable for RNA detection, quantification, purification by size, and quality assessment. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to </=20 nucleotides (nt). In certain circumstances, e.g., resolving different conformers of RNAs or RNA-protein complexes, native gels are appropriate. The only disadvantage to acrylamide gels is that they are not suitable for analyzing large RNAs (> or =600 nt); for such applications, agarose gels are preferred. This protocol describes how to prepare, load, and run polyacrylamide gels for RNA analysis.

Filter-binding assay for analysis of RNA-protein interactions.

One of the oldest and simplest (and still very useful) methods for detecting RNA-protein interactions is the filter-binding assay. If a mixture of RNA and protein is passed through a nitrocellulose filter, the protein will be retained and the RNA will pass through. But if the protein is capable of binding RNA, then RNA will be retained on the filter as well. This protocol requires a purified protein (or chromatographic fractions) of interest and (32)P-labeled RNA. To perform the assay, the protein sample is serially diluted to several concentrations. It is then mixed with a fixed amount of radioactive RNA and allowed to bind under desired conditions for 30-60 min. The binding reactions are then applied to a 96-well dot-blot apparatus with low vacuum to trap the complexes on three membranes: The top membrane traps aggregates, the middle membrane (nitrocellulose) binds proteins and RNA-protein complexes, and the bottom membrane (which is charged) collects free RNA. After washing and drying, the membranes are exposed to phosphor-imaging screens for quantitation. Alternatively, single, larger filters (that can be counted in a scintillation counter) and a filter manifold can be used.

Nondenaturing Agarose Gel Electrophoresis of RNA

INTRODUCTION Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. There are two common types of gel: polyacrylamide and agarose. For most applications involving RNAs of < or =600 nucleotides, denaturing acrylamide gels are most appropriate. In contrast, agarose gels are generally used to analyze RNAs of > or =600 nucleotides, and are especially useful for analysis of mRNAs (e.g., by Northern blotting). RNA analysis on agarose gels is essentially identical to DNA analysis (except that the gel boxes used must be dedicated to RNA work or to other ribonuclease-free work). Here we describe the use of straightforward Tris borate, EDTA (TBE) gels for routine analysis. These gels are appropriate for determining the quantity and integrity of RNA before using it for other applications. This procedure should not be used to determine size with accuracy, because the RNA will not remain in its extended state throughout the run.

Purification of RNA by SDS Solubilization and Phenol Extraction

This protocol describes a method for RNA purification by sodium dodecyl sulfate (SDS) solubilization and phenol extraction. It is of wide utility and is used routinely to deproteinize RNAs in biological material that has been solubilized in SDS, an ionic detergent that dissolves membranes, disrupts protein-nucleic acid interactions, and inactivates ribonucleases. Once solubilized, addition of phenol or phenol:chloroform:isoamyl alcohol (PCA) completely denatures the protein, and it becomes insoluble in aqueous solution. PCA extraction is the method of choice for preparing cytoplasmic RNA from tissue culture cells or in any other situation (e.g., enzyme reactions) where solubilization in SDS is easily achievable.

Reverse Transcription–Polymerase Chain Reaction

Reverse transcription coupled to the polymerase chain reaction (RT-PCR) is commonly used to detect the presence of mRNAs, pre-mRNAs, or other types of RNA such as noncoding RNAs. The method involves using a primer annealed to the RNA of interest. For mRNA, the primer is usually a synthetic oligo(dT)15-18, a random hexamer mixture (dN)6, or a synthetic DNA oligonucleotide that is complementary to a specific transcript (a gene-specific primer). This DNA:RNA hybrid serves as a template during reverse transcription, in which the enzyme reverse transcriptase (RT) generates a single-stranded cDNA copy of a portion of the target RNA molecule. Using random hexamer priming, it is possible to obtain representative cDNA copies of sequences from the entire length of the mRNAs and pre-mRNAs in a population. This cDNA can then be used as a template for PCR. On addition of gene-specific primers, a specific DNA fragment corresponding to a portion of the RNA of interest is generated.

Expression and Purification of Active Recombinant T7 RNA Polymerase from E. coli

For large-scale transcription reactions or for cost savings, a laboratory may want to prepare its own recombinant T7-, SP6-, or T3-phage RNA polymerases. It is convenient to perform this preparation every 2-3 years and have a consistent and reliable source of phage RNA polymerase for many in vitro transcription reactions. In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-d-1-thio-galactopyranoside (IPTG)-inducible promoter. The bacteria are lysed by sonication, the his6-tagged protein in the bacterial lysate is purified by binding to Ni-NTA agarose, and the resin is then extensively washed and eluted with imidazole. The purified enzyme is dialyzed against a glycerol-containing storage buffer and can then be stored for months or years at -20°C.

Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions

In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA.

Denaturation and Electrophoresis of RNA with Formaldehyde

Electrophoretic size fractionation can be used to denature and separate large mRNA molecules (0.5-10 kb) on formaldehyde-containing agarose gels. Formaldehyde contains a carbonyl group that reacts to form Schiff bases with the imino or amino groups of guanine, adenine, and cytosine. These covalent adducts prevent normal base pairing and maintain the RNA in a denatured state. Because these adducts are unstable, formaldehyde must be present in the gel to maintain the RNA in the denatured state. This protocol describes the preparation of an agarose gel with formaldehyde and its setup in a horizontal electrophoresis apparatus. RNA samples are prepared and denatured in a solution of formamide and formaldehyde and, with 0.5- to 10-kb size markers, subjected to electrophoresis through the gel. Following electrophoresis, the gel is stained to visualize RNA markers or rRNA using one of several different types of stains.