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Advancing the quality control methodology,,,to assess isolated total RNA,,,and generated fragmented cRNA

oth the bioanalyzer and the MOPS gels, some of them that were failed using the Agilent software were passed using gel electrophoresis, and vice versa. Some samples that were passed using the bioanalyzer and failed on the MOPS gel are shown in Figures 7 and 8.

Based upon the electropherograms, the acceptable quality of the samples is clearly determined, yet on the MOPS gel images a ribosomal band is missing from each. Just as the MOPS gel is capable of giving false negatives, it is also capable of giving false positives. Some examples of samples that were passed on the MOPS gel but failed on the bioanalyzer are shown in Figures 9 and 10. (Note: Samples that were passed using the MOPS gels were failed by QC measures further along in the process)


Based upon these observations, there are clearly more distinguishable results on the electropherograms produced using the bioanalyzer then there are in the MOPS gel images. There is a lot more room for human and mechanical error using the MOPS gels, which may contribute to the apparent differences.
Agilent 2100 bioanalyzer vs MOPS gels for fragmented cRNA
When analyzing fragmented cRNA, what is examined is that the sample has been allowed to heat long enough and is fully fragmented to a desirable level. The size of the fragments can be estimated using the ladder on the MOPS gel, but this is not conclusive and determination of fragmentation is very subjective due to the poor resolution of low molecular weight components (refer to Figures 1-3). The mRNA Smear Nano assay used with the bioanalyzer is capable of showing the desired fragment length range and percentage of the total area for each sample. This is an extremely valuable tool be
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