RNA visualized on the gels had similar quality, amount and RNA molecular weight between the starting sample and those following clean up. Humic acids in the starting sample absorbed at A230-320. Cleanup increased 260/280 and 260/230 ratios, consistent with pure RNA. Concentration of RNA following clean up decreased to an average of 129.93 ng/µl. Qubit® PicoGreen® Assay (not shown) confirmed quantification. As humic acids falsely inflated concentrations due to absorbance at A230-320, a decrease in concentration is expected from clean, purified RNA.
Examination of RNA described in Figure 1 using RT-qPCR and a Bacillus 16S assay compared 1 µl of undiluted RNA against a 1:10 dilution. Undiluted, purified samples were successfully amplified and approximately 3 cycles existed between the cleaned undiluted and 1:10 dilution, showing lack of inhibitors. Starting samples still contained inhibitors, as shown by a failure to amplify either the undiluted or 1:10 dilutions.