Transcriptome Profiling Support - Troubleshooting

GeneChip™ expression arrays generally have less than 2% false change. This result is determined by hybridizing a single sample to two arrays of the same type, and determining the percentage of probe sets that show a significant change. It has been shown that the false change rate from the arrays, as described previously, is significantly lower than the potential variabilities derived from different biological samples.

MAS5 requires the use of Mismatch probes which the HT PM Plate Arrays do not currently use. MAS5 can be used with the Advanced Configuration however the software will automatically shut down if analysis is run.

No, continue to finish products using the same procedures and products. New projects can be moved to PM only arrays.

• GeneChip™ Scanner 3000s that were shipped after September 15, 2003 with serial number 502xxxxx are enabled for high-resolution scanning.

• GeneChip™ Scanner 3000s that were shipped prior to September 15, 2003 with serial number 501xxxxx that have had the GeneChip™ Scanner 3000 High-Resolution Update installed by an instrument service engineer are enabled for high-resolution scanning. A sticker is applied to the rear of the scanner to indicate that the upgrade has been performed.

• Contact technical support to inquire about a scanner upgrade.

• GeneChip™ Scanner 3000 that were shipped after September 15, 2003 with serial number 502xxxxx are enabled for high-resolution scanning.

• GeneChip™ Scanner 3000 that were shipped prior to September 15, 2003 with serial number 501xxxxx that have had the GeneChip™ Scanner 3000 High-Resolution Update installed by an instrument service engineer are enabled for high-resolution scanning. A sticker is applied to the rear of the scanner to indicate that the upgrade has been performed.

• Contact your local technical support to inquire about a scanner upgrade.

Please contact Technical Support to obtain the high-resolution scanning patch.

At the time of the HG-U133 Set design the "_a" probe set was not defined. The non-unique probe set type, "_a", was introduced with the Mouse Expression Set 430 to indicate probe sets that recognize multiple alternative transcripts from the same gene. Probe sets with common probes among multiple transcripts from separate genes are annotated with a "_s" suffix. Another way to describe this is that "_a" probe sets have a gene cluster count = 1 and transcript count >1, and "_s" probe sets have a gene cluster count > 1 and transcript count >1. The gene cluster count and transcript counts are available through the NetAffx™ Analysis Center. For consistency, the names of existing probe sets with the "_s" suffices were not changed between the HG-U133 Set and the HG-U133 Plus 2.0 and HG-U133A 2.0 Arrays. The new content on the HG-U133 Plus 2.0 Array incorporates both the "_a" and "_s" probe sets.

AFFX-r2-Hs18SrRNA-5_at
AFFX-r2-Hs18SrRNA-M_x_at
AFFX-r2-Hs18SrRNA-3_s_at
AFFX-r2-Hs28SrRNA-5_at
AFFX-r2-Hs28SrRNA-M_at
AFFX-r2-Hs28SrRNA-3_at

The AFFX-r2-Hs18SrRNA and AFFX-r2-Hs28SrRNA probe sets (3', M and 5') were not included in the HG-U133 A 2.0 and HG-U133 Plus 2.0 array designs though they were included in the HG-U133 Set (A and B). The probe selection regions for 18SrRNA r1 and r2 probe sets are highly overlapping and have performed similarly in internal testing. There is less overlap between the r1 and r2 28SrRNA probe selection regions. Users who are interested in evaluating ribosomal RNA probe sets should use the AFFX-r1-Hs18SrRNA probe sets (3', M, and 5').

• GeneChip™ Scanner 3000s that were shipped after September 15, 2003 with serial number 502xxx are enabled for high-resolution scanning.

• GeneChip™ Scanner 3000s that were shipped prior to September 15, 2003 with serial number 501xxx that have had the GeneChip™ Scanner 3000 High-Resolution Update installed by an instrument service engineer are enabled for high-resolution scanning. A sticker is applied to the rear of the scanner to indicate that the upgrade has been performed.

• Contact technical support at techsupport@thermofisher.com to inquire about a scanner upgrade.

In many cases the probes do overlap quite heavily. Multiple probes, although overlapping, are utilized as they still provide separate physical measures of expression and add to the robustness of the platform. Care was taken to ensure that probes from the same probe set, however similar in sequence composition, are spatially separated on the array. In addition, the annotation library file contains information regarding how many independent probes there are, as well as the number of non-overlapping probes, in each probe set. Independent probes are defined as those that have less or equal to 13 base overlaps with other probes in the probe set. This information provides additional insight and may be helpful to users when interpreting unexpected results.

All annotation sources were considered. In cases where both strong support (i.e., aligned RefSeq mRNA sequences) and poor support (i.e., GENSCAN predictions) exist, both were used. Thus, a gene locus that contains a RefSeq mRNA will frequently contain probe sets representing not only the RefSeq exons, but also a number of other exons supported by other annotation sources. Slightly over half of the array is supported only by a single source and many of these are EST- or GENACAN- based annotations. See the Exon Array Design Technical Note for a more comprehensive discussion of the content on the array.

A significant amount of research was dedicated to developing optimal algorithms for generating the design to both ensure comprehensive coverage of putative exons and also prevent over-fragmentation. Many of these design criteria are discussed in the Exon Array Design Technical Note, such as restricting exon fragmentation to only those ESTs with consensus splice sites.