Agreement for AFFYMERIX
Transcriptome project
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue : XX.XX.XX
General information and conditions of access
To benefit from this support, interested teams will fill a request form to be sent to Sandrine
Balzergue (URGV) by email (balzerg@evry.inra.fr).
The target preparation, hybridization, scanning and analysis will be performed by dedicated staff of
the platform. The team brings in its expertise on transcriptome analysis and its workforce in this
collaboration but does not benefit from sufficient financial resources to support the cost of
consumables that will therefore be charged to the partner laboratory: 250 € HT per
hybridization + the price of the AFFYMETRIX chip.
A DVD containing every file related to the project, including raw and analyzed data, will be sent to
the collaborator. The experimental results will be integrated into the CATdb database (compatible
with standard MIAME: Brazma et al, 2001. Nat Genet. 29 (4) :365-71) and transmitted to the
Geomnibus database from NCBI for publication . You will be asked to fill an information form
about your project (From the biological question to your samples preparation).
The data will be released within 1 year after project completion; an email will notify you 15
days before. On the same date, the remaining RNA samples and amplified RNA will be
returned.
Technical data (protocols etc..) and a list of current collaborations are available on the URGV
website: http://www.versailles.inra.fr/urgv/microarray.htm
Affymetrix chip types available can be found on http://www.affymetrix.com/
Samples preparation
It is important to note that many factors influence the gene expression levels of a plant. The control of
experimental conditions is therefore crucial if we want to link a difference in expression to the
function studied. And a control plant compared to a plant having undergone a specific treatment
should be grown in the same light and nutritional environment as the latter. For example, a shift of
sampling time during the day will reveal differences due to the circadian expression of many genes. A
lack of homogeneity of watering or plant treatment can be a source of variability unrelated to the
studied process. These considerations should be taken into account to ensure the reproducibility of
sampling.
Replicate:
It is essential to distinguish between a technical repetition and biological repetition.
Technical replicate:
Technical repetitions of a sample are prepared at the same time (sowing, harvest, extraction ...).
Samples from different individuals but from the same experiment are considered as technical
replicates
Version du 07/24/2012
Page 1 / 3
Agreement for AFFYMERIX
Transcriptome project
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue : XX.XX.XX
It allows the observation and quantification of technical bias (technical variability), the control of the
reproducibility of the study and the quality control of the data but the results cannot be generalized.
Biological replicate :
Biological replicates samples are prepared in independent experiments (sowing, harvesting,
extraction ...) with at least a 24 hours shift (beware of the circadian cycle).
It allows the observation of inter-individual and inter-experiment variability.
The results can then be generalized.
It is necessary to provide at least one biological repetition, that is to say a repetition of the whole
experiment. The aim is to characterize the biological variability between replicates, and "remove" it,
to identify genes whose differential expression is related only to the studied factor.
As technical biases are negligible for this technology, we do not perform AFFYMETRIX technical
replicates.
Quantity and quality needed for experiments
A quantity of 4μg of total RNA per sample is to be expected with a minimum concentration of 100 ng
/ uL for chips 3 'and 50 ng / uL for chips WT protocol. The purity of RNA is one of the most
important factors for the success of the experiment; it is preferable to use a protocol column
extraction (RNeasy kit for example) including the step of DNase I. For "difficult" samples such as
seeds, the addition of PVP is very useful, contact us if needed. Total RNAs were sent in dry ice in the
elution solution. Their quality will be estimated on Agilent chip and will be quantified with
"RiboGreen" after arriving on the platform.
Total RNA will be sent together with the fully completed information table (see and print the last
page of this agreement).
Results publication
This is a project involving the collaborating scientists and the URGV. So, at least one member of
the URGV will be co-author of the first publication in which data obtained will be exploited.
You will also be asked to include in the description of the data, the CATdb database (example: "All
raw and normalized data are available-through the database CATdb (AFFY_XXXXXXX, Gagnot et
al, 2008) and from the Gene Expression omnibus (GEO) repository at the National Center for
Biotechnology Information (NCBI) (T. Barrett et al. NAR 2006): GSE accession number XXXXX. ")
Project Design (required):
We ask you to provide the following information:
1 - Title of Project
2 - Name and address of project manager
3 - Name and address of the person responsible for monitoring analysis in relationship with
URGV
4 - Scientific aims
5 – Experimental design ( sample 1 vs sample 2 ….)
6 – hybridization number – Description of sample per chip : species, organ, treatment …
Version du 07/24/2012
Page 2 / 3
Agreement for AFFYMERIX
Transcriptome project
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue : XX.XX.XX
7 - Expected date of delivery of samples to URGV:
-----------------------------------------------------------------------------------------------------------------------8 - Table to join the RNA samples when sending:
TUBE name
Version du 07/24/2012
Sample
name
experimental design
on Concentration µg/µl
RNA
extraction
method? DNAseI?
Page 3 / 3