In Vitro Synthesis of RNA
SP6, T7 and T3 phage RNA polymerases have high specificity for their respective 23 base promoters (Figure 1). The development of cloning vectors containing promoters for these polymerases has made the in vitro synthesis of single stranded RNA molecules a routine laboratory procedure. Modern multipurpose cloning vectors contain a multiple cloning site (MCS) flanked on each side by promoters for different polymerases. This allows the synthesis of either sense or antisense RNAs from sequences cloned into the multiple cloning site. Plasmid templates are generally linearized with a restriction enzyme to allow run off RNA transcripts to be synthesized with a defined end. In addition to plasmid DNA, PCR products and synthetic oligonucleotides can be used as templates for transcription reactions. For PCR products, one of the primers needs to include the promoter sequence of a phage polymerase so that the PCR product contains a phage promoter. Synthetic oligonucleotides need to contain the phage promoter, which must be double stranded. However, the remainder of the template need only be single stranded.
Figure 1. Consensus Promoter Sequences. The +1 base is the first base incorporated into RNA during transcription. The underline indicates the minimum sequence required for efficient transcription.
RNA Probe Synthesis with MAXIscript and MEGAscript Kits
RNA probes are frequently used for probing Northern and Southern blots because they can be synthesized at high specific activity; and since they are single stranded, they are not depleted by self-hybridization. In addition, RNA:RNA and RNA:DNA duplexes are more stable than DNA duplexes. RNA probes give lower levels of background signal with in situ hybridization, since background non-duplex probes can be degraded by RNase treatment. MAXIscript™ Kits are recommended for synthesis of radioactive and nonisotopic probes.
Synthesis of Large Mass Amounts of RNA - A Complete Selection of Kits
Ambion's high yield transcription kits are recommended for the synthesis of large mass amounts of RNA which may be used for in vitro translation, microinjection, ribozyme studies, microarray analysis, non-radioisotopic probes, and a variety of other applications. Ambion's original MEGAscript™ Kit can be used for all applications requiring high yields of RNA transcripts, including synthesis of capped RNA, and small RNAs whose yields are often very low with conventional reactions. However, while the original MEGAscript™ Kit allows the researcher to manipulate reaction conditions to optimize the reaction to a particular application, the kit is not supplied with specialized reagents such as cap analog or high concentration polymerase for synthesizing large amounts of small transcripts.
mMESSAGE mMACHINE™ Kits have been specifically designed for high yield synthesis of capped RNA for use in microinjection and in vitro translation studies. They utilize the same high yield technology as the MEGAscript Kits, and contain cap analog premixed with nucleotides in a single tube (4:1 Cap:GTP). The mMESSAGE mMACHINE Kit provides a simplified format for the routine synthesis of 15 to 35 µg of capped RNA per 20 µl reaction. Use the MEGAscript Kit when you need to manipulate the cap analog:GTP ratio. Cap analog is available separately for use in these applications.
Capped vs. Uncapped RNA Transcripts
Most eukaryotic mRNA molecules have a 5' 7-methyl guanosine residue or cap structure which both functions in the protein synthesis initiation process and serves to protect the mRNA from intracellular nuclease digestion. Capped in vitro transcripts can be synthesized by substituting cap analog (m7G(5')ppp(5')G) for a portion of the GTP in the transcription reactions. In vitro transcripts which are to be microinjected into oocytes or other cells, or used for transfection experiments or in vitro splicing reactions, should be capped. However, for in vitro translation experiments it is frequently not necessary to cap in vitro transcripts. Cap analog is a relatively expensive reagent and its inclusion in a transcription reaction often decreases the yield of the reaction. An alternative to capping in vitro transcripts is to use Ambion's Retic Lysate IVT™ translation kit, which is supplied with alternative buffers for translating uncapped in vitro transcripts.
The MEGAshortscript™ Kit is designed specifically to give high yields of small RNAs from short templates (< 300 bases). Synthesis from a given mass amount of RNA from a small template requires more initiation events than for a larger template. Since initiation is generally the rate-limiting step in an in vitro transcription reaction, the yields of small RNAs are normally relatively low. The MEGAshortscript Kit utilizes a high polymerase enzyme mix to maximize yields of small transcripts. MEGAshortscript Kits work well with oligonucleotide or PCR product templates as well as standard plasmid templates.
Which Ambion Kits to Use?
In vitro transcription reactions are generally used for two distinct purposes: the synthesis of high specific activity RNA probes, and the synthesis of larger mass amounts of RNA. MAXIscript Kits are recommended for the synthesis of high specific activity probes for use in ribonuclease protection assays, Northern and Southern blotting and in situ hybridizations. MAXIscript Kits can also be used for larger scale synthesis of RNA (5-10 µg/20 µl reaction) but are only about 1/10 as efficient as MEGAscript Kits for that purpose. A comparison of the yields of RNA from the MEGAscript Kit and conventional kit are shown in Figures 2 and 3. The very high yield of MEGAscript Kits is due to novel patented technology developed at Ambion. Ambion was the first company to introduce high-yield in vitro transcription kits. Reaction conditions have been optimized in MEGAscript Kits to allow the use of high concentrations of nucleotides which would normally be inhibitory to transcription. Thus, kits based on Ambion's new high yield technology are recommended for most applications requiring large scale synthesis of RNA. These include the MEGAscript, mMESSAGE mMACHINE, and MEGAshortscript kits.
Figure 2. Effect of Template Size on Yield of MEGAscript™ reactions. 20 µg reactions containing 1 µg of different DNA templates coding for the indicated sized transcripts were added to each reaction. Inset shows 0.25 µl aliquots of both conventional and MEGAscript™ reactions transcribing a 0.3 kb transcript separated on a 2% agarose gel and ethidium bromide stained.
Figure 3. mMESSAGE mMACHINE™ vs Conventional in Vitro Transcription Reactions for Synthesis of Capped Globin RNA. 20 µl transcription reactions containing 1 µg template. T7 mMESSAGE mMACHINE™ components and [³²P]UTP were incubated at 37°C. 1 µl aliquots were removed at 30-minute intervals and acid precipitable cpm determined by scintillation counting.
Does It Make Antisense?
When a DNA sequence is presented in scientific journals and databases, it is usually written as a single strand. By convention, the strand shown is the coding (+) or sense strand, identical in sequence (with T's instead of U's) to its mRNA copy. The mRNA then serves as a template for translation, its 5' or upstream sequence (beginning with AUG) corresponding to the NH3-terminal "Met" of the protein.
The complementary DNA strand is called the noncoding or (-) strand and corresponds to the antisense strand. In order to hybridize with and thus detect a specific mRNA, the probe must consist of complementary antisense sequence. Antisense RNA probes are generated when an RNA phage promoter adjacent to more downstream sequence is used.