The ability to synthesize RNA in the laboratory is critical to many techniques. Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions, can be used in blot hybridizations and nuclease protection assays. Such probes are much more sensitive than random-primed DNA probes. Small scale reactions may also be used to synthesize RNA transcripts containing modified nucleotides for various biochemical and molecular biology studies. Large scale transcription reactions, generating up to 200 µg of RNA per reaction can be used for aRNA amplification, expression studies (microinjection, infection with viral transcripts, in vitro translation), structural analysis (protein-RNA binding), and mechanistic studies (ribozyme analyses). In this article, we present an overview of transcription, including requirements of in vitro transcription reactions and a comparison of conventional vs. large scale RNA synthesis.
In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application. .
Rabbit Reticulocyte Lysate
Rabbit reticulocyte lysate is a highly efficient in vitro eukaryotic protein synthesis system used for translation of exogenous RNAs (either natural or generated in vitro). In vivo, reticulocytes are highly specialized cells primarily responsible for the synthesis of hemoglobin, which represents more than 90% of the protein made in the reticulocyte. These immature red cells have already lost their nuclei, but contain adequate mRNA, as well as complete translation machinery, for extensive globin synthesis. The endogenous globin mRNA can be eliminated by incubation with Ca2+-dependent micrococcal nuclease, which is later inactivated by chelation of the Ca2+ by EGTA. Ambion offers a nuclease-treated reticulocyte lysate. This type of lysate is the most widely used RNA-dependent cell-free system because of its low background and its efficient utilization of exogenous RNAs even at low concentrations (Figure 1). Exogenous proteins are synthesized at a rate close to that observed in intact reticulocyte cells.
Figure 1. Standard in Vitro Translation Procedure Using Rabbit Reticulocyte Lysate or Wheat Germ Extract.
Untreated reticulocyte lysate translates endogenous globin mRNA, exogenous RNAs, or both. This type of lysate is typically used for studying the translation machinery, e.g. studying the effects of inhibitors on globin translation. Both the untreated and treated rabbit reticulocyte lysates have low nuclease activity and are capable of synthesizing a large amount of full-length product. Both lysates are appropriate for the synthesis of larger proteins from either capped or uncapped RNAs (eukaryotic or viral).
The DNA template must contain a double-stranded promoter region where the phage polymerase binds and initiates RNA synthesis. Transcription templates include plasmid constructs engineered by cloning, cDNA templates generated by first- and second-strand synthesis from an RNA precursor (e.g., aRNA amplification), and linear templates generated by PCR or by annealing chemically synthesized oligonucleotides.
Many common plasmid cloning vectors include phage polymerase promoters. They often contain two distinct promoters, one on each side of the multiple cloning site, allowing transcription of either strand of an inserted sequence. Such dual opposable promoter vectors include Ambion's pDP, Promega's pGEM, Stratagene's pBluescript and Invitrogen's pCRII vectors.
Ambion's pTRIPLEscript family of vectors contain all three phage polymerase promoters in tandem (on the same side of the multiple cloning site), allowing any of the three polymerases, SP6, T7 or T3 to be used.
Plasmid vectors used as transcription templates should be linearized by restriction enzyme digestion. Because transcription proceeds to the end of the DNA template, linearization ensures that RNA transcripts of a defined length and sequence are generated. The restriction site need not be unique, and providing the promoter remains adjacent to the transcription template, the vector itself may be digested multiple times. It is also unnecessary to purify the promoter-insert sequence away from other fragments prior to transcription because only the fragment containing promoter sequence will serve as template. Restriction enzyme digestion should be followed by purification since contaminants in the digestion reaction may inhibit transcription.
PCR products can also function as templates for transcription. A promoter can be added to the PCR product by including the promoter sequence at the 5' end of either the forward or reverse PCR primer. These bases become double-stranded promoter sequence during the PCR reaction.
Two oligonucleotides can also be used to create short transcription templates. Two complementary oligonucleotides containing a phage promoter sequence are simply annealed to make a double-stranded DNA template. Only part of the DNA template — the -17 to +1 bases of the RNA polymerase promoter — needs to be double-stranded. It may be more economical, therefore, to synthesize one short and one long oligonucleotide, generating an asymmetric hybrid (see "Minimal Sequence Requirements").
A more recent use of in vitro transcription is in aRNA amplification reactions. For these reactions, transcription templates are generated from RNA by using an oligo(dT)-T7 promoter primer during reverse transcription. The cDNA is converted to a double-stranded transcription template by a second-strand synthesis reaction.
When designing a transcription template, it must be decided whether sense or antisense transcripts are needed. If the RNA is to be used as a probe for hybridization to messenger RNA (e.g. Northern blots, in situ hybridizations, and nuclease protection assays), complementary antisense transcripts are required. In contrast, sense strand transcripts are used when performing expression, structural or functional studies or when constructing a standard curve for RNA quantitation using an artificial sense strand RNA.
The +1 G of the RNA polymerase promoter sequence in the DNA template is the first base incorporated into the transcription product. To make sense RNA, the 5' end of the coding strand must be adjacent to or just downstream of, the +1 G of the promoter. For antisense RNA to be transcribed the 5' end of the noncoding strand must be adjacent to the +1 G. If the insert is in a vector, the vector should be linearized downstream from the promoter and the inserted sequence to be transcribed (see "Does It Make Antisense?").
In vitro transcription reactions can be divided into two types: conventional and large scale. Conventional reactions are typically used for synthesizing radiolabeled RNA probes or for incorporating modified nucleotides into transcripts. Large scale reactions, which generate >100 µg RNA per reaction, are useful for structural and expression studies, as well as for aRNA amplification.
Conventional Reactions: Synthesis of Labeled RNA Probes or Modified Transcripts
Conventional reaction conditions, such as those used in Ambion's MAXIscript™ Kit, use relatively low nucleotide concentrations (0.5 mM each). Higher nucleotide concentrations are not necessary since, in these reactions, the low concentration of radiolabeled or modified nucleotide present effectively limits the total yield of the reaction.
The total concentration of the limiting nucleotide (labeled/modified and unlabeled) should be at least 3 µM for efficient synthesis of full length RNA transcripts of <400 nt (more will be needed to synthesize longer transcripts).
A 3 µM concentration of radiolabeled rNTP can be obtained by adding 5 µl of a 800 Ci/mmol, 10 mCi/ml (or 12.5 µM) solution of [α-32P] NTP. Higher specific activity labeled rNTPs are available, but are provided at a much lower stock molar concentration (e.g. the 3000 Ci/mmol, 10 mCi/ml has a stock concentration of only 3.3 µM). Without the addition of unlabeled NTP, it is impossible to achieve the final minimum 3 µM reaction concentration.
Because limiting nucleotide concentration can result in premature termination of transcription, there is a trade-off between synthesis of high specific activity (or extensively modified) transcripts and full length transcripts. Diluting the limiting radiolabeled or modified nucleotide with unlabeled nucleotide proportionally lowers the specific activity (or extent of modification) of the transcript, but yields more full length transcript. To make very high specific activity or extensively modified transcripts one should limit or omit any unlabeled limiting nucleotide present.
When transcribing RNA from templates lacking CTP and TTP in the 12 bases immediately downstream from the transcription start site, the 3 µM limiting nucleotide minimum can be overcome (1). Ambion's CU Minus Promoter Technology provides vectors containing CTP and TTP-minus RNA polymerase promoters as well as conversion primers that can be used to eliminate CTP and TTP bases from RNA polymerase promoters in existing vectors. Such templates produce a high proportion of full length transcripts in reactions containing as little as 0.165 µM total limiting nucleotide. Using CU Minus technology, the highest specific activity radiolabeled nucleotides available can now be made by in vitro transcription without addition of unlabeled nucleotide. As a result, RNA probes with 7.5X higher specific activity can be transcribed.
Large Scale Synthesis: For Structural and Expression Studies, and aRNA Amplification
Large scale in vitro transcription reactions can produce up to 120-180 µg RNA per microgram template in a 20 µl reaction. Novel, patented technology developed by Ambion (i.e., MEGAscript™, see below) allows the phage RNA polymerases to remain active at high nucleotide concentrations that would ordinarily inhibit the enzyme. Yields from these large scale reactions are typically 10 to 50 times higher than those possible with conventional transcription reactions (without any limiting nucleotide). Reaction conditions (e.g. the type of nucleotide salt, type and concentration of salt in the transcription buffer, enzyme concentration and pH) are all optimized not only for each polymerase but for the entire set of components. Only under these conditions can you achieve optimal yields.
Ambion offers a complete line of products for in vitro transcription. The MAXIscript Kit is ideal for making radio- and nonisotopically labeled RNA probes for use in hybridizations. The probes generated by the Strip-EZ RNA Probe Synthesis and Removal Kits are readily stripped from Northern blots, enabling many rounds of hybridization without damaging nucleic acid bound to the blot.
The MEGAscript family of kits use Ambion's high yield patented technology to synthesize RNA for applications where large mass amounts are required. Large amounts of capped RNA transcripts can be synthesized with the mMESSAGE mMACHINE Kit using the same high yield patented technology. If desired, the Poly(A) Tailing Kit can be used to add a poly(A) tail to capped RNA transcripts synthesized with the mMESSAGE mMACHINE Kit.
The MessageAmp™ aRNA Kit is a complete kit for aRNA amplification based on the patented Eberwine method. Incorporating MEGAscript high yield transcription technology, this kit includes all necessary reagents for first-strand cDNA synthesis, RNase H digestion, second-strand synthesis, cDNA purification, in vitro transcription and aRNA purification.
- Ling M-L, Risman SS, Klement JF, McGraw N, McAllister WT. Abortive initiation by bacteriophage T3 and T7 polymerases under conditions of limiting substrate. Nucl. Acids Res. (1989) 17: 1605-1618.