Frequently Asked Questions

 

What is the minimum amount of total RNA I can use for each Affymetrix GeneChip expression array?

We currently recommend submitting 15ug of total RNA for each Affy experiment.  Although we use 5ug of total RNA for each eukaryotic expression the extra amount allows us to assess RNA quality and quantity. Reducing the amount of starting total RNA may result in a subsequent decrease in sensitivity. For those researchers with limited amounts of starting material, a protocl is available from Affymetrix for performing experiments using as little as 100ng of sample.

 

What method/protocol should I use to prepare total RNA for microarray analysis?

Although many methods exist for preparing total RNA, Affymetrix recommends RNA extraction using TRIzol reagent, followed by clean up with Qiagen RNeasy kits. Whichever method is used, the bottom line here is that pure, high quality RNA is needed to ensure good expression results.

 

What if I cannot isolate enough pure total RNA for the minimum requirement?

For researchers working with samples such as biopsies, laser capture microdissected tissue samples or flow-sorted cell samples where the amount of isolated total RNA is minimal, a protocol has been developed by Affymetrix which incorporates two rounds of cDNA amplification. According to the technical bulletin listed on Affymetrix’s website, good expression results can be generated from as little as 50-100ng of total RNA.

 

Can I hybridize samples to an array from a species other than the organism for which the array was designed? For example, can I hybridize a mouse sample to a rat chip?

Although there may exist a high degree of homology between closely related species, sufficient sequence differences may exist which could interfere with hybridization and data interpretation. Although some researchers have explored his approach, it is not recommended by our facility.

 

How can I get my data?

We can provide all data on CD/DVD upon request.  However, our web-based LINUX server allows researchers access to data files from their own PCs.  Simply by logging onto http://geneexpression.cinj.org (Or click on SERVER on the left) with a supplied username and password allows access their data.  Affymetrix files (.EXP, .DAT, .CEL, .CHP) and excel-based comparison files can be downloaded and stored or imported into third party analysis software.

 

I have just received my analysis data. What do I do next?

Data analysis can be the most important part of each expression experiment. Usually, Hierarchical and K-means clustering can be performed, provided that the researcher has run enough GeneChips. For instance, clustering should not be performed on a simple comparison analysis between experimental and control samples. In this situation, we recommend going to the NetAffx Analysis Center on Affymetrix's website. Here, gene expression data can be uploaded for structural and functional information, as well as gene ontology, for drawing biologically relevant conclusions.