UMR INRA 116 -UEVE-ERLCNRS 8196
General information on transcriptome
experiments with the “Complete Arabidopsis
Transcriptome Microarray”
CATMAv7
Validate by S. Balzergue date: XX.XX.XX
General informations and conditions
The CATMA microarray is available through collaborations between the URGV and other
laboratories. The partner needs to send a request to Sandrine BALZERGUE (URGV) via email (balzerg@evry.inra.fr).
The microarray production, and labelling and hybridizations of the samples will be performed by
the URGV team (Transcriptome Group). The URGV provides the know-how in this
collaboration but has no specific funding for that purpose. Thus the consumables costs
(280€ TF/hybridization) will be paid by the partner.
Specific tools for transcriptome analysis, including specific statistical methods are available at the
URGV. As soon as the analyses are performed, an Excel file contains transcriptomic results will
be sent to collaborator. Raw files can be sent to collaborator on request. Then the
transcriptome results will be integrated in the database developed at the URGV: CATdb
(MIAME compliant: Brazma et al., 2001. Nat Genet. 29(4):365-71). The data are also
transferred in GeOmnibus (NCBI). Be careful if you do not plan to publish all data at the same
time, fill 2 different files to get two accession numbers (if needed contact us for more
information.). Be careful if you do not plan to publish all data at the same time, fill 2 different
files for two accession numbers (if needed contact us for more information.)
The data will be released for public access one year after the end of the project and their
introduction into the databases. Nevertheless, there are exceptions that will be discussed before
the beginning of each project:
1) if it’s an industrial collaboration
2) if it is within the framework of an ANR / KBBE project and the results are placed in the
public one year after the end of the project itself only.
3) if the transcriptomic results has a value for filing patent
Note: A quote can be made and sent to collaborator only if requested to Sandrine Balzergue
(balzerg@evry.inra.fr). The collaborator must edit a purchase order in the amount specified by this
quote and in a delay of 15 days after receipt of samples. Collaborator must send the original order at
INRA-URGV, accounting department, 2 rue Gaston Cremieux 91000 Evry. If you want more
information, please contact: secretariat@evry.inra.fr
The CATMA microarray
We developed a new version of the CATMA microarrays based on AGILENT technology
(CATMA v7). In this new version, long primers instead of amplified GST are synthesized on the
array. We designed the array in order to keep as much as possible the same specificity to
Arabidopsis genes and to get (at least) the same coverage than the current version of the
CATMA array. A single high density microarray slide contains four chambers, each containing
180000 primers representing all the Arabidopsis genes: 38360 primers corresponding to TAIRv8
annotation (including primers of mitochondrial and chloroplast genes) + around 1200 primers
corresponding to EUGENE software predictions. Moreover, we included primers corresponding
to reapeat elements, for miRNA/MIR, and for other RNAs (rRNA,tRNA, snRNA, soRNA) and
finally 36 controls. Forward primer is triplicate in each chamber for robust analysis and Reverse
UMR INRA 116 -UEVE-ERLCNRS 8196
General information on transcriptome
experiments with the “Complete Arabidopsis
Transcriptome Microarray”
CATMAv7
Validate by S. Balzergue date: XX.XX.XX
sense primers are also available on a support.. With this new CATMA array, we can perform
more hybridizations at a same time, on a more stable support than the previous “in house
spotted” array and at slightly lower cost. A normalization and statistical analysis method has
been developed specifically for CATMAv7 by M.L. Martin-Magniette in collaboration with the
'Statistique et Génome' team from the unit UMR INRA/AgroParisTech MIA 518, Paris, France
Sample preparation
The labelling process includes an in-vitro transcription step (T7 pol.) so the RNA purity is the most
critical factor of the success. Therefore the RNA have to be extracted with a column extraction
kit (such as RNeasy for example with DNAseI step) . The samples will be controlled on the
Agilent bioanalyzer at the URGV. Generally, 4µg of total RNA per sample are sufficient for
running the experiment. If “difficult” samples, such as seeds for example, have to be used, please
contact us for extra precautions.
Repetitions
Biological repetitions: At least two independant biological replicates are necessary.
Technical repetitions : We perform systematically one dye-swap per biological replicate:
Ex:
- slide1: Control Cy3 – Treatment Cy5
- slide 2 : treatment Cy3 – Control Cy5
Procedure
The protocols are available at : http://www-urgv.versailles.inra.fr/microarray/protocols.htm
Summary of the protocol :
- Checking of the total RNA (Bioanalyser Agilent).
- Two-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp
Labeling (Agilent)
- Washing and drying of the slides.
- Scanning with constant PMT (photomultiplicators): the data normalization is performed at the
statistical analysis step.
After the statistical analysis the gene lists contain the log2 normalized intensities per samples, the
log2 normalized ratios, and the p-values (FDR correction) which provide the threshold for the
identification of the differentially expressed genes.
Publication of the results
In this scientific collaboration the URGV provides the know-how and expertise in transcriptome
analysis including statistics, and advices in experimental design if necessary.. Only the costs of
the chemicals are paid by the partner. For this reason at least one member of our team will be
co-author of the first paper reporting these transcriptome results. The same convention will
UMR INRA 116 -UEVE-ERLCNRS 8196
General information on transcriptome
experiments with the “Complete Arabidopsis
Transcriptome Microarray”
CATMAv7
Validate by S. Balzergue date: XX.XX.XX
be applied to the filing of Patent on the initiative of the collaborator and in which the
transcriptome results will be used.
Document to send with the request
We need a short document containing :
1 – PROJECT TITLE
2 – Name and address of the partner
4 – description the project (briefly)
5 – Experimental design
6 – Number of slides, samples description (organ, harvesting stage according to Boyes et al.
Plant Cell 2001, treatment…)
7 – Expected date of sample shipping
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