The PureYield™ RNA Midiprep System quickly and easily isolates high yields of pure total RNA from essentially any sample type and virtually eliminates copurification of DNA (1)
. The streamlined, universal protocol uses the PureYield™ silica-membrane technology to isolate intact RNA ranging in size from less than 200 bases to greater than 20kb. High-quality total RNA is isolated with minimal effort and can be used in a wide range of applications. The PureYield™ System eliminates the need for phenol:chloroform extractions and alcohol precipitations.
The RiboMAX™ Large Scale RNA Production Systems produce milligram amounts of RNA from 5–10µg of a linear DNA template. Transcripts up to 14kb have been generated; however, the RiboMAX™ System is more commonly used to generate transcripts up to 5–6kb in size. In vitro transcription reactions are widely used to synthesize microgram amounts of RNA probes from recombinant DNA templates. Most transcription reactions that are designed to generate RNA probes are optimized to maximize incorporation of radiolabeled ribonucleotides rather than to produce large amounts of RNA. However, in vitro transcription also is used for many other applications that require large amounts of biologically active RNA such as for producing tRNA, rRNA, small functional RNAs, RNA virus genomes, ribozymes and RNA for transfection.
In this report, we combine these two systems to purify milligram quantities of pure, highly concentrated RNA from RNAs synthesized using in vitro transcription reaction volumes of 100–1,000µl.
In Vitro Transcription
Seven in vitro transcription reactions were performed using the RiboMAX™ Large Scale RNA Production System—T7 (Cat.# P1300) following the protocol provided in the RiboMAX™ Large Scale RNA Production Systems—SP6 and T7 Technical Bulletin #TB166. A no-polymerase control was included with these reactions. Table 1 details the exact volumes used. For all reactions, the linear control DNA provided with the RiboMAX™ System was used to generate a 1,800bp RNA product. The reactions were incubated for 4 hours at 37°C. RQ1 RNase-Free DNase (Cat.# M6101) was added to the reactions according to Table 1, and the reactions were incubated at 37°C for 15 minutes. Ten microliters of each transcription reaction was saved for analysis on an agarose gel.
Table 1. Reaction Setup for the RiboMAX™ Large Scale RNA Production System.
In vitro transcription reactions were purified with the PureYield™ RNA Midiprep System (Cat.# Z3740) using a modified protocol from the PureYield™ RNA MidiPrep System Technical Manual #TM279. Table 2 details the volumes used; water was added first to bring the reactions to a final volume of 1ml.
Table 2. RNA Purification Setup Using the PureYield™ RNA Midiprep System.
The 1ml samples were combined with 1ml of RNA Lysis Buffer (with β-mercaptoethanol) in a 15ml conical tube. Four milliliters of RNA dilution buffer was added to each sample and vortexed to mix thoroughly. Isopropanol (4ml) was added and mixed by inverting the tubes. To capture the RNA, the entire mixture was then added to a clear PureYield™ Binding Column in a 50ml conical tube and centrifuged at 2,000 × g for 10 minutes in a swinging bucket rotor. After discarding the flowthrough, 20ml of RNA Wash Solution (with ethanol) was added to the binding column and centrifuged at 2,000 × g for 5 minutes. After discarding the wash buffer, 10ml of RNA Wash Solution was added to the column and centrifuged at 2,000 × g for 10 minutes. After discarding the wash solution, the column was centrifuged for another 5 minutes to help dry the membrane. The samples were eluted from the columns using an Eluator Vacuum Elution Device (Cat.# A1071). Water (600µl) was added to the Binding Column and incubated at room temperature for 2 minutes. A vacuum was applied to collect the eluate (Elution 1). For most of the samples, another 600µl of water was added to the Binding Column, incubated for 2 minutes and a second eluate was collected (Elution 2). For a subset of samples, Elution 1 was added back to the Binding Column, incubated for 2 minutes and recollected (Double Elution).
Eluates were analyzed for RNA concentration and purity using spectrophotometry according to the NanoDrop® ND-1000 manual. The transcription reactions and post-purification eluates were also analyzed on a 1.2% agarose gel. One microliter of each sample was mixed with 17µl of formaldehyde sample buffer (Lonza Cat.# 50571) and 2µl ethidium bromide (0.01µg/µl final concentration). Samples were heated to 70°C for 5 minutes, and 20µl was loaded. The gel was run at 100V for 30 minutes, and RNA was detected using a Bio-Rad XR imager with ethidium bromide filter.
In vitro transcription reactions were purified using the PureYield™ RNA Midiprep System as described in the Methods section. RNA concentration and purity ratios were evaluated using spectrophotometry (Table 3). All eluates had detectable RNA, except for the minus T7 polymerase control, which had no detectable nucleic acid (not shown). RNA concentration increased as the transcription reaction (TRN) volume increased. The quantity of RNA purified was linear over the 100–1,000µl reaction volume range (Figure 1). For all samples, significant RNA concentrations were detected in the second elution, sometimes as much as 80% of Elution 1. The highest concentrations were observed when performing a double elution, where Elution 1 was added back to the column and eluted a second time. This method resulted in yields of 3mg and 5.7mg for the 500µl and 1,000µl reactions, respectively, with concentrations of 5.6µg/µl and 11.4µg/µl. The total amount of RNA in the elution is linear from 100–1,000µl, indicating the maximum binding capacity of the binding column was not reached. A260/A280 and A260/A230 ratios were around 2.0, indicating highly pure nucleic acid.
Table 3. RNA Eluates Analyzed Using the NanoDrop® ND-1000 Spectrophotometer.
Samples were also analyzed on an agarose gel as described in the Methods section (Figure 2). One microliter of each sample (transcription reaction before PureYield™ purification and eluate after purification) was loaded onto a 1.2% agarose gel. The amount of sample loaded as a percent of the total is indicated in Figure 2. Only the predicted 1,800 base product was seen. Based on gel analysis of the transcription reactions prior to purification, the DNase reaction was complete (no 3,500bp DNA template observed). The RNA recovery was excellent because the amount of RNA in the eluate appeared to be proportionally the same as the amount in the transcription reaction. A direct comparison of input RNA and final eluate is shown in the red box in Figure 2; the 500µl input sample (lane labeled T) has the same band intensity as the 500µl eluate, indicating good recovery from the PureYield™ Binding Column. We also observed no significant degradation of the RNA, demonstrating the samples are clear of ribonucleases.
The PureYield™ RNA Midiprep System is capable of purifying RNA synthesized by in vitro transcription. As the reaction volumes increased, RNA yield and concentration also increased. The purified RNA was highly pure with little degradation based on absorbance ratios and gel analysis. To maximize RNA recovery, a second elution is recommended; the first eluate may be reapplied to the column to maximize RNA concentration. Based on gel analysis comparison, there is a substantial RNA recovery using this method. We recommend using one PureYield™ Midiprep Binding Column per ≤1ml of in vitro transcription reaction (e.g., a 4ml reaction would require four PureYield™ columns).