We have prepared cDNA microarrays to be used to scan for gene expression differences between two RNA sets. We would like to scan through a large number of RNA sets to establish a gene expression database showing how genes are regulated under different conditions. We know that we are getting very good reproducibility from chip to chip (10-30% difference in RNA ratios from the same RNA batch).
We have recently made the full genome chip, with help from Steve Jones at the Sanger center. The full genome chip has a 1 kb piece of exon rich genomic DNA for every gene.
We encourage isolating RNAs from synchronized, stage specific worms. In a mixed stage population, RNA regulation at one developmental stage is diluted by constitutive expression at other stages. We know that the biggest difficulty is showing which expression differences are reproducible from prep to prep, and which are not.
We encourage sending us gain-of-function mutants in addition to loss-of-function and wild-type. We will do all pair-wise combinations, but the gf versus lf combination may be the most sensitive.
Please email Stuart (email@example.com) a list of RNAs and a tentative time schedule so that we can anticipate the number of experiments. We can make a large number of chips and so can theoretically hybridize a large number of RNA sets.
We will hybridize any RNAs that you send us (time permitting). If different labs send us the same RNAs, we will hybridize both sets.
Specifications for RNA to be used for microarray analysis:
-5-10 micrograms of once-selected polyA-RNA from each source are
required for each hybridization.
-Optimally, we would like 15-30 micrograms of polyA-RNA so that we can do each experiment in triplicate.
-Optimally, we would like 3 independent samples of RNA from each source (i.e. 3 x 30 micrograms of polyA RNA total of each source) to show reproducibility.
-We would like a detailed description of the source of RNA, namely the growth conditions and isolation procedures.
-The RNA should be shipped frozen in ddH2O on dry ice at a concentration of about 500 nanograms/microliter to 1 microgram/microliter. TE is unnecessary. Alternatively, the RNA may be shipped as a dry pellet on dry ice.
We can make about 500 chips from one PCR, 50 chips in one day, and can hybridize and scan several RNAs in three days.
We will immediately send you the data from your RNA. Your data will be private for 6 months. After 6 months, other labs that do worm chip experiments will be able to view your data to compare it to their own. After the 6 month waiting period, we feel strongly that this data be semi-public since one inherent advantage of the chip is that the same genes are being analyzed under many different conditions. Once you have a set of target genes, it will be important to know how each target is regulated under different conditions. The data will be public once it is published.
We are interested in using the chip to globally analyze gene expression differences among dozens or hundreds of different conditions. We want to identify gene batteries, to compare sets of downstream targets, to determine whether pathways are convergent, linear or divergent etc., and to compare RNA differences from new conditions to those from well established and well understood conditions.Return to the Kim lab home page