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.
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.