Maximizing The Yield Of Full-Length RNA From An In Vitro Transcription Reaction

Ron Meis, Jim Pease and Katharine Kramer

Abstract

The quality and quantity of RNA produced in an in vitro transcription reaction depends upon a number of factors, including RNA transcript size, template concentration, reaction time and temperature. These parameters can be modified to optimize the yield of full-length RNA from the transcription reaction. In an individual application, the template used, and the resulting transcript, determine how the reaction should be modified. Here we present ways to maximize the RNA yield for both long and short transcripts using a commercial in vitro transcription kit.

Introduction

Generally, in vitro transcription reactions include a T7, T3, or SP6 RNA polymerase. These enzymes were originally discovered in their respective bacteriophage and were subsequently cloned to conveniently provide sufficient quantities of highly purified enzymes.^(1,2) Each polymerase recognizes a short (20 required bases), well-defined, phage promoter sequence with a high degree of specificity, making the enzymes very useful reagents for in vitro transcription.^(3,4)Conventional in vitro transcription reactions include rNTPs (0.5 mM each nucleotide), reaction buffer, linear DNA template (1 to 2 μg) and the appropriate bacteriophage RNA polymerase. These reactions produce 10 to 40 μg of RNA and can be used to prepare RNA for techniques that do not require large quantities of RNA, such as labeled RNA probes.

Increasingly important techniques, such as gene expression profiling with microarrays, RNA interference gene-silencing, in vitro translation, ribozymes, and RNA structure studies, require larger quantities of RNA.⁽⁵⁾ To address this need companies have developed commercial, high-yield in vitro transcription kits. Commercial in vitro transcription kits add various, proprietary components to their transcription reactions, which enable the polymerase to transcribe in the presence of 10 to 20 fold higher concentrations of rNTPs. These reactions use 1 μg of DNA template to produce 100 to 200 μg of RNA.

Commercial kits optimize reaction conditions to maximize the RNA yield from control templates, typically producing 1 to 2 kb transcripts, but each user's unique transcript may benefit from adjustments to the kit protocol. By varying reaction conditions such as the template concentration, temperature and time, it is possible to maximize the quantity of RNA transcribed from a specific template. Here we present data showing the results of changing these parameters to transcribe both long (>500; bases) and short (<500; bases) transcripts.

Many commercial in vitro transcription kits are available. For this review we chose to investigate reaction parameters using the AmpliScribe™ T7-Flash™ Transcription Kit (Epicentre, Madison, Wis.), which combines high RNA yield with fast reaction time (30 minutes). Generally, the results of reaction variations presented here should apply to other commercial in vitro transcription kits, although specific RNA yields and reaction times may vary significantly.

Template preparation

The DNA template used for in vitro transcription should be linear, double-stranded, and relatively clean. Typical templates include linearized plasmids, with blunt or 5'-protruding ends, PCR products or cDNAs, which were generated by adding a phage promoter sequence to the 5' end of a primer,⁽⁶⁾ or double-stranded oligonucleotides. Template should be free of RNase and contaminants, such as phenol, trace metals and sodium dodecyl sulfate (SDS). Usually, if the DNA can be digested by restriction enzymes, it will be clean enough to use as a transcription template. Treating the DNA with proteinase K, followed by phenol/chloroform extraction and ethanol precipitation usually results in a sufficiently clean template.⁽⁶⁾ Confirm that the template is fully linearized and intact by analyzing the DNA on an agarose or polyacrylamide gel and staining with ethidium bromide or SYBR®-Gold; (Molecular Probes, Inc., Eugene, Ore.).

Maximizing yields of long (>500; kb) RNA transcripts

Template concentration and reaction time for a long transcript

A high concentration of template DNA is less critical in a long-transcript reaction than in a short-transcript reaction. Increasing the template concentration reduces the reaction time, rather than increasing the RNA yield. For example an AmpliScribe T7-Flash reaction, containing 1 μg of linear 4.2-kb plasmid template DNA for a 1.4 kb transcript, produces the maximum RNA yield of 160 to180 μg in 30 minutes. Increasing the amount of template to 2 μg reduces the reaction time to 15 minutes for the same RNA yield.

Optimizing long transcripts from limited amounts of template

For some applications, the amount of template DNA is limited (less than 1 μg). In these cases, increasing the reaction time or temperature, or scaling up the reaction, can maximize the RNA yield. Figure 1 shows the RNA yield for a 1.4 kb transcript using 100, 50 and 10 ng of template DNA, over time. With 100 ng of template DNA the reaction went to completion by 2.5 hours. The 10 ng reaction never reached the maximum attainable RNA yield, even after 4 hours. However, if a limited amount of template is available, a significant amount of RNA can be made by allowing the reaction to incubate for 2.5 to 3 hours.

Reaction temperature for a long transcript

Increasing the temperature from 37 C to 42 C for the long-transcript reaction not only increased the rate at which the RNA was produced, but also increased the maximum attainable RNA yield, as shown in Figure 2. The effect of increasing the reaction temperature is more significant in reactions with lower template DNA concentrations (10 ng). With lower concentrations of template, the long-transcript reaction at 37 C never reached the RNA yields produced by the 42 C reaction. With a limited amount of template, it may be advantageous to run the reaction at 42 C for 2.5 to 4 hours.

Scale-up reactions for long transcripts

AmpliScribe T7-Flash reactions can be scaled up to produce milligram quantities of long RNA transcripts. By scaling up all components, including the template, by 6 to 10 fold, these reactions can produce milligrams of RNA in 30 minutes. If template is limited, a scaled-up reaction will still significantly improve the RNA yield. For example, a 10 fold scaled-up reaction containing just 10 ng of template DNA produced 230 μg of RNA in an overnight reaction (data not shown). This represents a 23,000-fold increase in RNA yield over template concentration.

Maximizing yields of short (<500; bases) RNA transcriptsBecause initiation is the rate-limiting step in a transcription reaction, the dynamics in a short-transcript reaction differ from those in a long-transcript reaction. When producing a short transcript, a greater percentage of the nucleotides are incorporated during the slower, initiation step than during the faster, elongation step. (i.e. 8 nucleotides of the 26 total nucleotides are incorporated during initiation). Since fewer nucleotides are incorporated into final product per initiation, many more initiations are required to approach the maximum RNA yield for the reaction. Therefore, the best way to optimize transcription of short RNAs is to maximize the number of possible transcription initiations, by increasing template concentration or reaction times. With optimized reaction conditions, the number of moles of RNA produced in a short-transcript reaction frequently exceeds the number of moles of RNA produced in a long-transcript reaction by at least several fold, as shown in Table 1.

Template size and concentration for a short transcript

Increasing the template concentration in an in vitro transcription reaction increases the RNA yield of a short transcript, as shown in Figure 3. In addition, when transcribing a short RNA, the size of the template needs to be considered. Generally, in vitro transcription reactions use 1 μg of template DNA. By comparison, 1 μg of a 75 bp double-stranded oligonucleotide template is 20 pmoles of DNA, whereas 1 μg of a 4.2 kb linear plasmid is only 360 fmoles of DNA. Accordingly, 1 μg of an oligonucleotide template produces more short RNA than 1 μg of a plasmid template in otherwise equal reactions.

Reaction time for a short transcript

When transcribing a short RNA, increasing the reaction time will increase the number of transcription initiation events and significantly increase the RNA yield. For example, incubating the AmpliScribe T7-Flash reaction for 3 to 4 hours provides a significant, 2 to 5 fold, increase in the RNA yield compared to the standard 30-minute reaction. After 3 to 4 hours the yield starts to plateau (Figure 3).

Reaction temperature for a short transcript

Typically in vitro transcription reactions incubate at 37 C. However experiments show an increase in RNA yield and shorter reaction times, when reactions are incubated at 42 C. With 1 μg of template DNA, incubating the reaction at 42 C can significantly reduce the amount of time needed to obtain the maximum yield (Figure 4). The 42 C reaction temperature does not affect the RNA yield as significantly at higher template concentrations.

Conclusion

RNA yields for both long and short transcripts from in vitro transcription reactions can be maximized by adjusting the template DNA concentration, the reaction time, and the reaction temperature. For long transcripts, limited DNA template concentrations can produce significant quantities of RNA by incubating the reactions for 2 to 4 hours at 42 C. Scaled-up reactions readily produce milligram amounts of long transcripts.For short transcripts, increasing the DNA template concentration gives the most significant improvement in the RNA yield. Increasing the reaction time and/or increasing the reaction temperature from 37 C to 42 C also increases the RNA yield.

About the authors

Ron Meis (Epicentre) generated the data presented here. Jim Pease, and Katharine Kramer (Epicentre) prepared the manuscript. The authors thank Julie Capadona (Epicentre) for graphic design work.

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