Affymetrix Users

Affymetrix Data:

• Take a look at methods used in target preparation/hybridization and initial data analysis.
• Find out how to retrieve data for your Affymetrix transcript profiling experiment.

Manuals:
Download manuals for Affymetrix products, including:

• NetAffx Analysis Center User's Guide (pdf, 1 MB)
• Expression Analysis Technical Manual (pdf, 5.38 MB) includes detailed protocols for expression analysis including sample preparation, target labeling, hybridization, washing scanning and basic data analysis.
• Microarray Suite User's Guide, Version 5.0 (pdf, 361 KB) includes details on how to use MAS to control the GeneChip Fluidics Station and Scanner, analyze experimental data and generate reports that summarize intensity data, analysis output and algorithm settings.
• And many others.

NetAffx:
The NetAffx™ Analysis Center enables researchers to correlate their GeneChip® array results with array design and annotation information. This online resource provides the scientific community with access to array content information, including probe sequences and gene annotations.

Microarray Suite Version 5.0:

• Review the technical note of the MAS 5.0 algorithms
• Review Statistical Algorithms Reference Guide

Additional Resources:

• Scientific Publications allows researchers to search the publications using Affymetrix technology. The full list is also available.
• Grant Writing Resources provides descriptions about GeneChip technology and products to streamline your grant-writing process.
• Technical Notes regarding GeneChip Arrays and software.

The size of the PCR product from the ApoE gene is 270 bp in length. For E2, there is no resultant HhaI digestion, resulting in bands 91 bp and 83 bp in length. For E3, the HhaI enzyme cuts only at one site, resulting in bands 91 bp, 48 bp, and 35 bp in length. E4 has the same restriction sight as E3 as well as one within the 91 bp fragment region, resulting in bands 72 bp, 48 bp, and 35 bp in length. Since the nucleotide substitutions in the three common alleles alter only two HhaI cleavage sites, all genotypic combinations can be distinguished with the combination of duplicate or any two of the genotype bands described above.

Once the informative STR (short tandem repeat) allele for a pair of donor and recipient is established, that particular loci is used to track the ratio of donor and recipient cells after post donor lymphocyte infusion. For the post samples, a positive result is determined by the percentage of donor allele present in that specimen.

The expected Vector PCR product band size is 647 bp and the expected Avigen Control Plasmid PCR product is 547 bp. Each spiked reaction should have the specific 547bp control plasmid band. A specimen is interpreted as negative if the 547 bp control band is present and the 647 bp vector band is not present. A specimen is interpreted as inhibited if both the 547 bp control band and the 647 bp vector band are not present. A specimen is interpreted as positive for vector if the 647 bp vector band is present.

A form of identity testing which clarifies familial relationships, STR (short tandem repeat) loci consist of short, repetitive sequence elements of 3 to 7 base pairs in length. These abundant repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which often may be detected using polymerase chain reaction. Alleles of these loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and distinguished from one another using fluorescence detection following electrophoretic separation.

This screening can be used for a qualitative assessment of clonal diversity among peripheral blood T cells, T-cell lines and clones emerging from bulk and limiting dilution cultures. It can also be used as a tool for assessing the clonal composition of T-cell populations. In some cases, it is desirable that a culture system propagates T cells with uniform efficiency (as seen in the example).

Nomenclature of data analyzed

Master Mix Set					Size
1	2	3	4	5
>3	>--	13	16	22	378+15
2	6	12	--	23	329+15
4	8	11	18	25	276+15
5	9	14	17	24	222+15
1	7	15	20	21	170+15

Specimens are tested in “Process Groups,” with each process group consisting of 380 specimens and 4 controls (XX, XY, YY and no DNA controls); the number of process groups will depend on the number of specimens submitted for testing.

The results for your SNP genotyping assay(s) tested on a set of controls and on your study specimens are reported to you in several formats.

A scatter plot (as a jpg file readable in Adobe Photoshop) for each individual SNP and each process group (if more than 380 samples analyzed). Controls are shown in aqua color. The three genotypes (XX, XY and YY are shown in red, green and blue, respectively. Black X’s indicate uninterpretable results.

Individual text files of SNP genotyping data for each process group (380 sample per process group), including Excel file of Well number, Sample ID, SNP name (marker name), X signal and Y signal intensities, Genotype (call), Quality Value, how genotype was called (Call Type: Automatic or Manual) in columns.

One Report Summary for each process group in an Excel file that has been reviewed by the director. Each Excel file has three tabbed sections:

• “SNP Report” that summarizes all SNP Genotype results for one process group.
• “ABI Data” that details all SNP genotypes for one process group.
• “Genotypes” showing a summary statistical analysis by specimen (for all SNPs analyzed) and by SNP (for all specimens tested). “FF” indicates an undetermined result. An empty cell (no result for a SNP) indicates a result removed prior to analysis of the rest of the data since the result was distant from the other genotype clusters.

Controls

Each SNP assay is tested on a set of control DNAs and these results are reported separately from your study specimens. The control DNAs consist of African-American and Caucasian populations and one CEPH family (three generations of a single family with known pedigree). The control results determine how the assay is working, allele frequencies, accuracy of genotype calling, and if all three genotypes are present (classic, bigenotypic or monogenotypic). The CEPH family results are reviewed using a PEDCHECK program to assess the accuracy of the genotype calls.

We choose controls from this assay and use it while running the specimens. Three control specimens showing the three genotypes (XX, XY and YY) are selected and used as controls when the study specimens are tested.

If the control run shows monogenotypic pattern we contact the investigator and do not run the specimens.