Xine volume 3, issue 4
Dear Colleagues:
Welcome to Xine, the source for Xenopus news and information. Here's what's happening.
There is lots of news on genomic resources for Xenopus and a request for comments
from the community.
As always, you can read a hypertext version of Xine here
http://blumberg-lab.bio.uci.edu/xine/index.htm
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http://blumberg.bio.uci.edu/xine/index.htm
or here
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From Rob Grainger and Richard Harland
To the Xenopus research community:
On October 21 & 22 a group will be meeting at NIH to discuss needs for community-wide
genomic and genetic resources for Xenopus research. At an NIH-sponsored meeting in 2000,
guidelines were established for projects now underway, including sequencing of ESTs,
construction and sequencing of cDNA libraries, funding of genetic resources, construction
and sequencing of BAC libraries and whole-genome sequencing. The full list of priorities
from 2000 is listed in the 2000 meeting report:
http://www.nih.gov/science/models/xenopus/reports/xenopus_report.pdf
Because that report was written over three years ago, and considerable progress has
occurred in our field, it is once again time to set priorities for community-wide
resources. Toward that end we strongly encourage you to write to us with your suggestions
for NIH-sponsored initiatives that you feel are important for the Xenopus community.
Please review the goals in the 2000 report (page 5, attached), commenting on goals
that should be maintained or modified, as well as suggestion for new goals. Send your
comments to: rmglab@virginia.edu by October 10.
Sincerely,
Rob Grainger and Richard Harland
Information on the Xenopus microarray chips
that are being made by Affymetrix
General information from Affymetrix:
1. Who is making the array?
A Xenopus Consortium worked with Affymetrix to create the array.
2. What will be on the array?
There was a call for sequences to be deposited into GenBank by May 2003. A UniGene build
was assembled (June 2003, build 36) and utilized by Affymetrix to seed a clustering and
assembly process. This process culminated in the creation of a consensus sequence to
represent a potential transcript isoform.
Cluster quality was assessed as an objective means to rank sequences for representation,
e.g. EST stacks within the assembly, polyA sites and signals, biological evidence for
expression, sequence annotation, and sequence orientation. Not all of the UniGene
clusters, particularly EST singletons and doubletons, will be represented.
In general, the same design process used for Affymetrix catalog products,HG-U133, Rat 230,
and Mouse 430, were applied. A point of note is that probe selection for the Affymetrix
gene expression assay is biased towards the 3' end of transcripts. More details about the
design process and probe selection can be obtained from the current human, mouse and rat
design "technotes" available on the Affymetrix website.
3. What is an Affymetrix probe set and how are these chosen?
Gene transcripts are represented by probe sets that consist of multiple 25mer
oligonucleotides. Oligonucleotides are synthesized in situ on the array. Independent
measurements provided by multiple probes are required for specificity and sensitivity. A
probe selection algorithm consisting of heuristic and model based rules is used. Model
based rules predict linear dose response profiles as well as the potential for cross
hybridization. Two basic types of probe sets are created: 1) those that represent gene
sequences uniquely, 2) those that represent multiple closely related gene sequences.
Affymetrix probe sets typically consist of 11 to 16 probe pairs. For the Xenopus array 16
probe pairs were selected per probe set. A probe pair consists of a 25mer perfect match
oligo and a reference 25mer mismatch oligo. For the eukaryotic gene expression assay,
labeled antisense cRNA target is hybridized to the complementary 25mer probes.
4. How do I get information about the Affymetrix assay?
A thorough description and diagram of the assay can be found on Affymetrix.com. Briefly:
the starting material is either total RNA (minimum of 5 µg) or poly-A mRNA (minimum of
0.2 µg). The sample is reverse transcribed, using a T7-oligo(dT) primer, and
double-stranded cDNA is synthesized. The double-stranded cDNA, with the incorporated T7
promoter, is then used as a template in the subsequent in vitro transcription reaction.
Biotinylated UTP and CTP are incorporated into cRNA during the in vitro transcription
reaction. Labeled cRNA is detected via streptavidin-phycoerythrin staining.
5. Where (How) do I run my experiments?
Affymetrix arrays must be processed on GeneChip® Systems (processing is also available in
institutional core labs, or service providers for those who do not have systems installed
locally in department or individual labs).
6. Are annotations and support available?
Annotations will be available via the public NetAffx database at Affymetrix.com.
Annotations will be updated on roughly a quarterly basis.
7. When will it be ready?
The chip design (i.e., the content) has been completed. The goal is to have a GeneChip®
probe arrays available by December of 2003.
8. How much will it cost per chip and per experiment?
Chip prices are volume-based and are set based on institutional agreements. Academic
pricing will be no more than the current Mouse 430 GeneChip. Please check with your local
Affymetrix representative who can assist with more accurate pricing based on your
institution's agreement.
A general comment on the cost per experiment: In almost all cases, the biological
variation is substantially larger than the variation caused by chip itself. To determine
what level of variation there is, a pilot experiment is recommend consisting of 3 controls
and 3 treatments to determine how much statistical power is required.
9. How many gene products will be represented?
The Xenopus laevis GeneChip® will contain 15,500 probe sets representing approximately
11,911 gene clusters and 14,387 gene transcripts. To insure proper representation, a gene
or gene transcript may be represented by more than one probe set.
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Until next time.
Bruce
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