Examination of Global 3' Bias on Microarrays
|Users of Affymetrix GeneChip® probe arrays often monitor 3' to 5' (3'/5') ratios of housekeeping genes to control for sample quality and amplification reaction efficiencies (see sidebar, "What is the 3' to 5' Ratio?"). Similar quality checks are performed during the manufacturing process of Applied Biosystems Ambion® MessageAmp™ products. The newest addition to the product line, the MessageAmp Premier Kit, is no exception. In addition to a number of other specific metrics, the quality control procedure requires that aRNA prepared using the kit meets rigorous 3'/5' signal ratios when hybridized to the GeneChip human U133A2.0 probe array, before it can be sold as an Applied Biosystems product. These array-based functional tests for quality control are among the most stringent in the industry.|
Unexpected 3'/5' Signal Ratios From the ß-actin Probe Set
Figure 1. Low RNA Input May Result in Higher 3'/5' Ratios for b-actin. Human Brain Reference RNA (HBRR, Applied Biosystems) and Universal Human Reference RNA (UHRR, Stratagene) were used in RNA amplification and labeling reactions with either the Ambion® MessageAmp™ Premier Kit (50 or 500 ng RNA input amount) or the Affymetrix GeneChip® One-Cycle Kit (1000 ng RNA input amount) according to respective protocols. Array results were read and ß-actin 3'/5' ratios were calculated using GCOS software. Note that more RNA is required for the Affymetrix kit protocol than for the MessageAmp Premier Kit. JMP software was used to build plots.
Global Assessment of 3' Bias Confirms that GAPDH is a Better Indicator than ß-actin
Figure 2. 3'/5' Signal Ratios for All Probe Sets. Partek GS software was used to extract probe level signals for all probe sets with n = 11 probes (RMA Background subtraction was applied without quantile normalization). 3'/5' ratios were calculated by simply dividing the 3'-most probe by the 5'-most probe for each probe set. The ratios for all probe sets were then represented as standard box plots.
Kelli S. Bramlett, Penn Whitley, Charmaine San Jose, Natalie Sununpong Hernandez, Bob Setterquist • Applied Biosystems Inc., Austin, TX