Introduction
NASC checks the RNA quality at two points. We check the RNA quality when we first receive the samples from our users to make sure that there has been no degradation in transit. We then check again at the cRNA stage before fragmentation and hybridisation, to save wasting chips if the amplification and labelling process has not worked
NASC uses the Agilent Bioanalyzer to check the integrity of RNA. The output from the Bioanalyzer produces a graph that serves a similar purpose as a RNA gel. Along the X-axis measures time, which translates to fragment size. The shorter the time, the smaller the RNA fragment. The Y-axis measures the amount of RNA at that size (which would be the equivalent to darker bands in the gel). A 25bp RNA fragment is always spiked in to every sample- this always appears at the 24 second mark in the graphs below.
The ladder below shows the principle of size separation and quantitation by time.
We also use a NanoDrop to calculate concentrations of the initial sample and to check the 260/280 ratio as an assessment of nucleic acid purity. A ratio of ~2.0 is generally accepted as 'pure' for RNA.
Total RNA from Users
Qiagen
A typical profile for a high quality RNA prep using the Qiagen Kit is represented below.
The size of the 18S peak and 28S peaks are calculated. The 28S/18S ratio is one of the key indicators of RNA quality - a ratio of 2 is ideal.
Trizol
We also accept Trizol extracted total RNA. The quality of RNA extracted using the Trizol reagent is not as clean as with the Qiagen Kit. However, a greater mass ( >100mg ) of tissue can be treated with Trizol to obtain higher RNA yields than with the Qiagen kit.
Notice the band at 25 seconds. This is the 5S ribosomal RNA and tRNA, and not small degraded RNA. The Trizol prep usually results in samples with good 5S ribosomal band isolation, in comparison to Qiagen column purification in which the 5S and tRNA are too small to be isolated along with the ribosomal and messenger RNA.
Degraded
The electropherogram below is typical of a degraded total RNA.
Notice the raised baseline between the marker peak and the two ribosomal peaks. As a sample degrades, the mRNA between the ribosomal bands will appear to rise while the 28S ribosomal band shrinks and becomes broader. A broad band at 22-24 seconds may also start to appear. This is where the highly degraded RNA migrates. The electropherogram for this degraded sample is slightly shifted to the left.
Unfragmented cRNA
The quality of the biotin labelled unfragmented cRNA is checked before GeneChip analysis. A poor cRNA prep usually indicates poor quality starting RNA.
The electropherogram for this unfragmented cRNA has been overlaid with the ladder to show the general size distribution of the cRNA. The ladder fragments are: 200, 500, 1000, 2000, 4000 and 6000 nt in length. A cRNA profile with a greater proportion of fragments above 500 nt is usually considered as a good prep. A 28S/18S ratio of zero indicates that there is no ribosomal RNA contamination.