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Millipore Technical Publications

Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters

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Introduction

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies.

Centricon and Microcon centrifugal filters are well suited for the purification of radiolabeled RNA transcripts1. Ultrafiltration can simultaneously and efficiently remove unincorporated ribonucleotides and salts from the transcripts and concentrate the RNA. RNA molecules retain their integrity and are recovered with high yields.

Purity of a transcript is especially important when it is used in in vitro translation systems. Trace amounts of ethanol, phenol, salts or excess cap analog used during the synthesis of capped mRNA can cause a dramatic decrease in translation efficiency.

After the transcription reaction is complete, template DNA is usually degraded by the addition of Dnase I. The RNA is purified by two phenol/ chloroform extractions followed by ethanol precipitation. Other, less popular methods are gel purification (used predominantly when separation of full-length transcript from shorter RNAs is important, e.g., ribonuclease protection assays) or LiCl precipitation.

A series of experiments was performed in our laboratory to determine the effectiveness of using Centricon and Microcon devices to purify in vitro synthesized mRNA and in vitro translation studies. Results indicate that ultrafiltration can efficiently remove inhibitory contaminants from mRNA preparations, leading to increased translational efficiencies.

Methods: RNA Transcription
For our studies we chose plasmid pGEM-luc containing the luciferase gene (luc) in the center of a multiple cloning cassette of the pGEM-11Zf (-) plasmid (Promega). DNA template was linearized with XhoI, followed by enzyme and salt removal by diafiltration in Microcon 100K NMWL devices.

Linearized template was transcribed, using MEGAscript kit (Ambion) according to the recommended protocol. After the reaction was completed (3 to 4 hours), template DNA was degraded with Dnase I and the reaction mix added to a Microcon 30K NMWL device filled with 450 µL of water. The device was spun for 20 minutes at 12,000 x g in a temperature-controlled centrifuge at 4 °C.

Purified, concentrated RNA was recovered by inverted spin. For the preparation of capped transcript, cap analog m7G (5’) ppp (5’) G (New England Biolabs, Inc.) was included in the transcription reaction and the level of GTP reduced (4:1 ratio of cap analog to GTP). To purify the transcript by phenol/chloroform extraction, the reaction mix was diluted with water and a one-tenth volume of ammonium acetate stop solution was added. The mixture was extracted once with phenol/chloroform, followed by chloroform extraction. RNA was precipitated with isopropanol and the pellet resuspended in distilled water. Alternatively, LiCl precipitation solution (one-half volume) was added to the reaction mix, followed by incubation at minus 20 °C for 1 hour. RNA was pelleted by centrifugation and dissolved in water. Size and integrity of the in vitro transcription products were assessed by running an aliquot of the purified RNA transcript on a formaldehyde/formamide agarose gel. Ethidium bromide was added to the RNA before lading on the gel to stain the RNA sample and keep background fluorescence low2.

Translation In Vitro
In vitro translations were performed in the Flexi™ Rabbit Reticulate Lysate System (Promega) according to standard luciferase RNA translation conditions with minor modifications (Rnasin Ribonuclease inhibitor was omitted and 35S-methionine added). Results of translation were analyzed by determination of percent incorporation of 35S-methionine and fold stimulation, compared to controls without RNA. Minimum acceptable stimulation was 8-fold.

Results
Aliquots of RNA transcript purified by different methods (ultrafiltration, phenol extraction and LiCl precipitation) were run on a denaturing agarose/formaldehyde gel. Results are shown in Figure 1.1. The banding pattern of the 1.7kb RNA transcripts is identical regardless of purification method. Similar results were obtained in the case of capped transcript (results not shown).

The effect of increasing the mRNA concentration on the translational efficiencies was examined. At low mRNA levels, the capped luc mRNA was translated three times more efficiently than the uncapped mRNA (Figure 1.2). At higher mRNA levels, the translation of both transcripts was comparable. Similar behavior was observed with CAT mRNA3. Even relatively high levels of mRNA did not cause the decrease in translational efficiencies noted by other groups4. This result could be attributed in part to the lack of inhibitory contaminants in the mRNA preparation.

We also checked the effect of the RNA clean-up method on in vitro translational efficiency. For details of various procedures, see the Methods section. RNA purified by each of the methods (1 µg) was translated and results showing total 35S-methionine incorporation are presented in Table 1.1. While there were no observable differences between these RNAs by gel electrophoresis analysis (Figure 1.1), RNA purified by ultrafiltration gave twice the translation efficiencies of phenol-extracted RNA.


Figure 1.1 (left) Comparison of pGem-luc transcript purification methods. Transcripts were synthesized in 20 µL reactions. After Dnase I treatment, RNA was purified from the reaction mix. 500 ng of purified RNA were run on 1% agarose/formaldehyde gel.

Figure 1.2 (right) Effect of pGem-luc RNA concentration on in vitro translation. Increasing amounts of uncapped Luc RNA transcripts and capped Luc RNA were used in translation reactions. Incorporation of 35S-methionine was determined by TCA precipitation. Both RNA transcripts were purified with Microcon 30K NMWL devices.

Table 1.1 Effects of RNA clean-up method on translation efficiency
RNA
Total 35S-methionine Incorporation
RNA 1
972,000
RNA 2
514,000
RNA 3
776,000

The RNA transcripts were cleaned using Dnase I incubation followed by purification in Centricon 100K NMWL devices (RNA 1), phenol/chloroform extraction (RNA 2), or precipitation with 7.5 M LiCl (RNA 3). 1 µg of each RNA was used to program 50 µL in vitro translation reaction, using the Flexi Rabbit Reticulocyte Lysate System. 35S-methionine incorporation was determined by TCA precipitation. Data show the average of three independent experiments.

References
  1. Krowczynska AM. BioSolutions 1993;2(1):1–2.
  2. Ogretmen B, Ratajczak H, Kats A, Stark BC. Biotechniques 1993;14(6):932-5.
  3. Polayes D. Focus 1991;13(4):130 –2.
  4. Dasso MC, Jackson RJ. Nucl. Acid. Res. 1989;17:3129.