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Product FAQ

What is the maximum number of freeze/thaw cycles recommended for the CytoScan Reagent kit?

Answer

The CytoScan Reagent kit has been validated for less than or equal to 5 freeze/thaw cycles.

Answer Id: E14279

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Product FAQ

What is the difference between GeneChip miRNA 4.0 Cartridge Array, miRNA 4.1 Array Strip, and Affymetrix miRNA 4.1 Array Plate?

Answer

The arrays have the exact same design and contain the same number of probes and probe sets.

Answer Id: E14120

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Product FAQ

What procedure is used to generate the genome alignment data?

Answer

Genome alignments are currently provided for Human/Mouse/Rat/Drosophila/C elegans arrays. We align the target sequences against the genome sequence downloaded from the UCSC website using BLAT. While some of the target sequences do not align, perhaps due to the draft nature of several genomes, some targets align at multiple locations on the genome. We apply a filter to select the best hit for each target sequence.
We use the following procedure:

Calculate a score for each alignment as follows:

score=matches - (mismatches+5*qbaseinsert)
where matches = number of bases that match (including both repeat and non-repeat regions)
mismatches = number of bases that do not match in the alignment
qbaseinsert = number of bases inserted in the query

It is therefore possible that some of the scores are negative.

Pcgood=score*100/target size

The pcgood metric is provided on the website and in the download files.

Select the alignment with the best score.

Derive genomic coordinates for the probes (25-mers) from the "best" target sequence alignment.

We use the genomic coordinates for each probe (25-mer) from above and search for transcripts (RefSeq and GenBank mRNA alignments to the genome from UCSC genome database) that overlap with the alignment of the probes. In the NetAffx summary report, we provide the transcript whose genomic alignment overlaps with the maximum number of probes from that particular probe set. We also provide the total number of probes from the probe set that overlap with the transcript as measure of the ability of the probe set to detect the corresponding transcript.

Answer Id: E13479

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Product FAQ

How many SNP and CN probes are included on CytoScan Optima Array?

Answer

CytoScan Optima Array has a total of 315,608 features covering control, CN, and SNP probes. There are a total of 18,018 non polymorphic CN probes and 148,450 SNP markers on the Optima Suite.

Answer Id: E13840

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Product FAQ

How can I test if blocking of cDNA synthesis is specific for globin mRNA transcripts isolated using the GeneChip Globin-Reduction Kit?

Answer

The specificity of the method can be tested by using the GeneChip Globin Reduction RNA Controls kit which contains two control samples, Jurkat RNA and Jurkat RNA + Globin RNA. These controls mimic blood RNA samples not affected by excess globin message (for example, an erythrocyte lysis fractionation method) and affected by excess globin (for example, a PAXgene blood RNA sample) respectively. Besides providing additional possibilities for validation of the globin reduction procedure, target preparation starting from Jurkat RNA (which is free of globin transcripts) can be performed with and without integrated globin reduction.

Thus, specificity can be confirmed by comparing the expression profiles obtained from each sample. If non-globin transcripts are unaffected by the globin reduction procedure, no significant differences in the expression profiles derived is expected. For more information on the GeneChip Globin Reduction RNA Controls kit, please refer to the GeneChip Globin-Reduction Kit handbook (http://media.affymetrix.com/support/downloads/manuals/globin_reduction_protocol_manual.pdf).

Answer Id: E13488

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Product FAQ

What has been used as reference RNA for FlashTag labeling?

Answer

Ambion FirstChoice Total RNA samples have been used as reference RNA.

Answer Id: E14057

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Product FAQ

Why is the bgrd_mean (background) value sometimes higher than that of PM_mean (signal)?

Answer

The mean of the background probe signal values is based on background probes defined in the background probe file, which are by default the anti-genomic probes. Anti-genomic probes consist of about 1,000 probes for each level of GC content (0 to 25) with no homology to most studied organisms. This set has a higher GC content than the average probe on the array, and therefore can have relatively higher signal values than the mean of all probes (PM_mean).

Answer Id: E14027

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Product FAQ

When performing the CytoScan Assay, what can cause faint or absent bands on the PCR gel?

Answer

Several things can cause this problem. To troubleshoot this problem, first determine if the positive control worked properly. Common reasons for this failure include incomplete digestion of genomic DNA or inefficient ligation of adaptors, ligation samples that are not properly diluted or mixed, and degraded DNA (if only the positive control worked). See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038) for more information

Answer Id: E13966

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Product FAQ

What are the 10 CytoScan assay processing steps listed in order?

Answer

The 10 assay processing steps, as listed in the CytoScan Assay User Manual (Cat. No. 703038) are the following:

1. Genomic DNA Preparation
2. NSP1 Restriction Enzyme Digestion
3. Ligation
4. PCR
5. PCR Product Purification
6. Quantitation
7. Fragmentation
8. Labeling
9. Hybridization
10. Wash, Stain, Scanning

Answer Id: E13962

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Product FAQ

What 3'/5' ratio for control genes, for example GAPDH and Actin, should I anticipate to obtain on GeneChip probe arrays?

Answer

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 also been selected for certain housekeeping genes, including GAPDH and Actin. Signal intensity ratio of the 3' probe set over the 5' probe set is often referred to as the 3'/5' ratio. This ratio gives an indication of the integrity of your starting RNA, efficiency of first strand cDNA synthesis, and/or in vitro transcription of cRNA. 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.

There is no single threshold cutoff to assess sample quality for all of the diverse organisms and tissues. This is due to the presence of different isoforms of these house-keeping genes and their different expression patterns in various tissues and organisms. Although we routinely refer to a threshold ratio of less than 3 for the most common tissues, such as mammalian liver and brain, this may not be applicable to all situations. It may be more appropriate to document the 3'/5' ratios within a particular study and flag the results that deviate, therefore representing an unusual sample that deserves further investigation.

Answer Id: E13597

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Product FAQ

In the Command Console Software, what is sample/array registration?

Answer

It is the process of associating a sample record to a physical array or set of physical arrays. With this feature, users can link a sample to a single array, a sample to an array set (such as the GeneChip HG-U133A and B Arrays, or the GeneChip Human Mapping 500K Array Set), or a sample to an array type in replicate (one sample run on multiple arrays of the same type).

Answer Id: E14243

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Product FAQ

How many single nucleotide polymorphism (SNP) and copy number (CN) probes are included on the CytoScan HD Array?

Answer

CytoScan HD Array includes a total of approximately 6.5 million probes: 1 probe per allele in triplicate for 750,000 SNPs probes and 1.9 million non-polymorphic probes.

Answer Id: E13834

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Product FAQ

What quality control tools are available for the Axiom Genome-Wide BOS 1 Array?

Answer

Dish QC (DQC) is the recommended QC metric for Axiom Genome-Wide Arrays in Genotyping Console Software. The default threshold is greater than or equal to 0.82 for each sample. It operates by measuring signal at a collection of sites in the genome that are known not to vary from one individual to the next. Because the metric monitors non-polymorphic locations, at each position it is known which of the two channels in the assay should contain signal and which should be just background. DQC is a measure of the extent to which the distribution of signal values is separated from background values, with 0 indicating no separation and 1 indicating perfect separation.

Answer Id: E14044

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Product FAQ

What does the "_s_at" extension represent in the HG-U133 probe set name?

Answer

The primary goal in probe set selection is to select a probe set unique to a single transcript or common among a small set of similar transcript variants. A probe set name is appended with the "_s_at" extension when all the probes exactly match multiple transcripts. The probe set selection process generally favors probe sets measuring fewer transcripts. Probe sets with common probes among multiple transcripts (the "_s_at" probe sets), are frequent and are to be expected, due to alternative polyadenylation and alternative splicing. In most cases, "_s_at" probe sets represent transcripts from the same gene, but the same probe set can sometimes also represent transcripts from homologous genes. One transcript may be represented by both a unique and an "_s_at" probe set when the transcript variation is sufficient.

Answer Id: E14178

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Product FAQ

How should the thermal cycler settings be programmed for use with the CytoScan 750 array?

Answer

  Applied Biosystems Veriti 96-well Bio-Rad DNA Engine PTC-200 Eppendorf Mastercycler Pro S
Lid temperature 103 degrees C 103 degrees C 103 degrees C
Temperature mode N/A Calculated Safe
TSP heated lid N/A N/A Yes
Switch off lid at a low block temperature N/A N/A No

Answer Id: E14193

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