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DNA Array Core Facility

Quality Control

The DNA Array Core Facility instills various quality control methods throughout the RNA sample processing. When samples are first submitted, they are analyzed on the NanoDrop ND-1000 UV-Vis Spectrophotometer, providing the concentration and 260/280 values of the submitted sample(s). Approximately 1ug of each sample is then run on an agarose electrophoresis gel in order to assess the potential degradation of the total RNA. Samples with insufficient quantities for this gel analysis will be run on the Agilent Bioanalyzer. The Bioanalyzer uses as little as 50-500ng RNA to achieve a complete sample quality assessment. Upon request, any submitted sample may be run on the Bioanalyzer for an additional fee. Please contact the Core Facility at (858) 784-2263 for more information.

Electropherograms generated by the Agilent Bioanalyzer

Figure 1. Electropherograms generated by the Agilent Bioanalyzer showing (A) high quality, (B) degraded and (C) partially degraded total RNA samples. Each trace represents 50ng of total RNA and data are scaled to match peak heights between traces. (The two distinctive peaks in figure A represent the ribosomal RNA)

Samples that pass the initial quality control checkpoint, mentioned above, will automatically be processed using the appropriate RNA protocol. If any problems are noted regarding this initial quality control, the Investigator will be contacted as soon as possible.

Additional agarose gels are run at various points throughout the RNA sample processing protocols in order to assess the quality of the samples as they are labeled and fragmented. Once the quality of each sample is assessed and approved, it is hybridized to the appropriate GeneChip.

Photo of Agarose gel of RNA runs

Figure 2. A)Agarose gel of total RNA run with Low DNA Mass Ladder at 90V for 30 minutes, showing distinct 18S and 28S ribosomal RNA bands. B) Agarose gel of biotin-labeled cRNA run with Low DNA Mass Ladder at 90V for 30 minutes, showing a broad smear of material. C) Agarose gel of fragmented biotin-labeled cRNA run with Low DNA Mass Ladder at 90V for 30 minutes, showing transcript sizes <100bp.

Included in the hybridization cocktail are Affymetrix Eukaryotic Hybridization Controls, used to monitor and troubleshoot the hybridization process. The DNA Array Core Facility notes the presence of these controls on our sample tracking sheets when the GeneChips are scanned. Also noted on the sample tracking sheets are the background, noise, percent present, GAPDH ratio, and Actin ratio. These values are also used to assess the quality of the hybridization, and are available for viewing on our website query. Please note that this site is password-protected. To obtain a password for your lab, please contact Jennifer Hammond, or call the Core Facility at (858) 784-2263.

For further information or questions regarding quality control, please contact the Core Facility at (858) 784-2263.