FGSR Quality Control/Quantity Assessment

Because microarrays are so sensitive to RNA degradation, all samples submitted for microarray work are first analyzed for quality and quantity. By requiring this check at the beginning, we can simplify data analysis (by removing poor performers early) and help investigators potentially save a lot of money

RNA

Unless the PI specifically requests otherwise (in the Special Instructions of the submission sheet), the following steps will be performed as part of RNA QC/QA:

  1. The sample is quantified in 10mM Tris on the Nanodrop ND-1000 Spectrophotometer. In addition to concentration, this instrument reports the 260/280 absorption ratio, which can be an important indicator of hidden contaminants.
  2. The sample is then analyzed for integrity using either an Agilent 2100 Bioanalyzer or a Caliper LabChip GX. These instruments produce an electropherogram that makes it easy to spot certain contaminants or degradation, and to calculate the 28s:18s ratio.

Both the Agilent and Caliper instruments assign an integrity score to RNA samples - Agilent calls it an RNA Integrity Number (RIN), and Caliper an RNA Quality Score (RQS). These scores both lie on the same scale, and can be interpreted in a similar way. For both, a score of 1 represents complete degradation, while 10 represents an intact sample. For microarray work, we recommend that all samples have a minimum score of at least 7.0

The RIN and RQS are superior to other single measures of quality (such as the 28s:18s ratio) because they consider the entire electropherogram, rather than just one or two points. There are occasions, however, where the software refuses to assign a score. This does not mean that your RNA is bad! In this case, we advise investigators to examine the electropherogram, 260/280 ratio, and 28s:18s ratio to make a quality determination. If you need help with such a case, please contact us.

DNA

DNA submitted for a Genotyping project does not undergo a separate QC step - investigators should ensure the purity and cleanliness of their sample before submission. The FGSR does accept DNA for QA/QC as part of Quality Control projects, however. Unless a specific request is made in the Special Instructions of the order, the sample concentrations will be measured using Invitrogen PicoGreen Assay which measures only double stranded DNA. Concentrations measured using PicoGreen assay are usually lower than measurements on the Nanodrop. To ensure that samples are meeting minimum concentration requirements, FGSR recommends checking samples using the PicoGreen assay before shipment to FGSR.