The following information is important for those who are interested in using our Affymetrix microarray system:
The minimum requirements for the new Affymetrix Express Kit are 100ng of high-quality total RNA, in no more than 3ul (per GeneChip). In addition, we need 50ng or 1ul (whichever is larger) for quality measurement on the Bioanalyzer. The sample concentration can be measured on our Nanodrop or our Qubit fluorometer.
Massive amounts of total RNA are lost when samples are frozen for even short periods of time in regular RNase-free microcentrifuge tubes. For this reason, we suggest that total RNA be provided in non-stick microcentrifuge tubes, such as Ambion Cat# 12350.
- In order to reduce variation between samples, we try to prepare all samples simultaneously until hybridization. For this reason, we request all samples be provided at the same time.
Affymetrix recommends using a RNA isolation protocol that has been established for the tissues or cells being used. In the absence of an established protocol, using one of the commercially available kits designed for RNA isolation is suggested. Please see additional information in Section 2 of the "GeneChip Expression Analysis technical manual", below.
Important: If going directly from TRIzol-isolated total RNA to cDNA synthesis, it is beneficial to perform a second cleanup on the total RNA before starting. After the ethanol precipitation step in the TRIzol extraction procedure, perform a cleanup using the QIAGEN RNeasy Mini Kit. Much better yields of labeled cRNA are obtained from the in vitro transcription/ labeling reaction when this second cleanup is performed.
- Basic QC is done by comparing the intensity of the background, housekeeping controls, and gradient controls between chips. This is done both to determine quality of the scan itself and consistency between multiple scans. Some variation is expected between chips, but too much variation may indicate problems.
- Should a hybridization fail due to chip failure, Affymetrix will usually replace the chip at no cost. As the hybridization mix can be reused multiple times, a chip failure will cause a delay, but usually no additional cost. A chip problem is usually indicated by one chip giving bad results; if all the chips give bad results, that's usually a sample or prep problem, and won't be replaced by Affymetrix. If Affymetrix does not replace the chips, you will be charged the full amount, even if the data does not meet your needs.
The data is released via Rosetta Resolver, which is an analysis program that runs only under Windows. You can download the software here: http://gec.caltech.edu/ and you can get an account by emailing David Mathog (firstname.lastname@example.org) or Vijaya Rao (email@example.com).
- If you need to compare new data to older data labeled using the One Cycle kit, we do have a small amount of that kit type left; it requires at least 2ug of total RNA.