Gene Array Quality Control - Explanation


Four quality checking parameters were derived by the UTMB molecular genomics laboratory. There are no set cutoffs for any of these measurements. One factor alone isn’t a good indicator of the quality of data. When all of the parameters as a group appear to be deranged, one can conclude that the quality of the results may have been influenced by degradation of the RNA.

  1. Scaling factor: SF stands for scaling factor (SF). A low SF is good and a SF of 1 is the most desirable value. A low single digit value is generally considered acceptable, but there is no set value.
  2. Background average: BG Avg stands for background average.  A low value is good. There is no set maximum background. It is the experience of the laboratory that a BG average less that 100 leads to the cleanest data.
  3. Percent Present: This value represents how many of the probesets on the chip were judged to be expressed in the sample. The value varies from species to species and tissues.  Human chips in the 40s are considered acceptable.
  4. 3'/5' ratio: This is a 3’ to 5’ ratio for GAPDH. Signal intensity ratio of the 3' probe set over the 5' probe set is often referred to as the "3'/5' ratio." It is an indication of the integrity of the starting RNA, and/or the efficiency of first strand cDNA synthesis, and/or the in vitro transcription of cRNA. This value is one indicator of how degraded a sample might be. There is no single threshold cutoff to assess sample quality for all of the diverse organisms and tissues. Each sample type has distinct isoforms of these house-keeping genes and has different expression patterns. A threshold ratio of less than 3 for the most common tissues is considered adequate (such as mammalian liver and brain). One method that is potentially effective to spot degradation effects on hybridization results is to flag the 3'/5' ratios in a particular study that deviate strongly from the rest of the values.
Added information: In addition to the conventional probe sets designed to be within the most 3' 600 bp of a transcript, additional probe sets in the 5' region and middle portion (M) of the transcript have been selected for certain housekeeping genes, including GAPDH and Actin). Also referred to as HS-AFFX-HUMGAPDH/M33197-SIG35. The signal of each probe set reflects the sequence of the probes and their hybridization properties. A 1:1 molar ratio of the 3' to 5' transcript regions will not necessarily give a signal ratio of 1.