Large-Scale Transcriptome Analysis
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1. Scaling factor (SF): In creating our .CHP file, we used an all-probes scaling setting of 150 on each array. The SF for each array should be compared and should be approximately equivalent. For example, if the SF of one array is 1.215 and the second array has a SF of 1.561, these arrays overall are of approximately equal intensity. If one array’s SF is 1.335 and the second is 26.321, they can’t be compared with any confidence. 2. Background: The average background on the array should be less than 100. All arrays in an experimental set should have similar values. 3. Noise (RawQ): The noise is a reflection of the normal operation of the machine and should be less than 10. 4. Total probe sets: The percentages of number present should be in a range of 5–10 % of each other. Depending on the experimental conditions, the set of control samples and the set of experimental samples should each be similar among like samples. 5. Housekeeping controls: The housekeeping controls are representative of successful processing of the sample as the source of these genes are naturally occurring genes present in the sample. The housekeeping genes have intensity readings for probes from both the 3′ and 5′ ends and can be used to generate a 3′ to 5′ ratio value. If full mRNA templates were present in the sample, the ratio representing both ends of the gene that was processed through the amplification steps in the preparation of the sample should theoretically be equal to 1. If the starting mRNA was degraded and processed, this ratio would vary as the 3′ end of the mRNA would not be present for amplification. In our experiments, GAPDH (rat) was used as the housekeeping gene, and the acceptance value for the 3′/5′ ratio was 3 or less. Other common housekeeping genes on the Affymetrix rat genome array include beta-actin and hexokinase. 6. Spike controls: These controls are the Affymetrix Eukaryotic Hybridization Controls (mixed biotin-labeled bioB, bioC, bioD, and cre, in staggered amounts) incorporated as part of the hybridization cocktail. The bioC, bioD, and cre should all have present calls for both the 3′ and 5′ regions. The bioB is the control that is spiked in the least amount to test the lower limit of the readability by the scanner and has three regions that are investigated (3′, 5′, and middle). It is recommended that at least two of the three regions have present calls. Once the above visual evaluations and report standards have been met, the arrays are ready for analysis of the entire data set. The .CEL files for each array can be transferred out of either the GCOS or AGCC software and uploaded into various third-party statistical packages for the mining of significant results.
