Notes:The following is important for those who are interested in using our Affymetrix microarray system:
- The quality of the RNA is essential to the overall success of the analysis. Since the most appropriate protocol for the isolation of RNA can be source dependent, Affymetrix recommends using a 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.
- We currently use the One-Cycle cDNA Prep Kit and have found no significant problems. However, it is important to start with at least 2ug of total RNA. suspended in 8ul in order to produce an adequate amount of IVT RNA. See the general notes page to read about the loss of RNA when stored in regular RNase-free microcentrifuge tubes.
- Fragmentation prior to hybridization is imperative, therefore we request that you allow us to perform this step, even if you do not choose to have us process the RNA. In the rare case that the fragmented IVT RNA does not show an adequate electropherogram when run on the Bioanalyzer, the experiment will be halted until the source of error can be determined. This is done in order to save significant time and money.
- In order to reduce variation between samples, we intend to prepare all samples simultaneously up until the fragmentation step. For this reason, we request all samples be provided at the same time.
- After the GeneChips are scanned we investigate the intensity of the background, housekeeping controls, and gradient controls. This is done both to determine quality of the scan itself and consistency between multiple scans. Some variation is expected between chips. However, rarely is this variation significant between replicates or even samples from the same experiment.
- The GeneChips must not be cleaned using 70% ethanol. This disrupts the seal on the chip screen and can lead to leakage.
- Chips may be scanned multiple times. However, each additional scan or partial scan results in a slightly lower intensity reading. This can lead to unintended variation between replicates. For this reason, we try to perform as few scans as possible.
- For additional information on the GeneChip technology and methods, and for FAQs lists, please visit: http://www.affymetrix.com/support.index.affx
Additional up-to-date information on our Affymetrix system can be found on these wiki pages: http://jumpgate.caltech.edu/wiki/Affymetrix
Methods and ProtocolsGeneChip Expression Analysis Technical Manual
Data Analysis Fundamentals
Affymetrix Eukaryotic Sample and Array Processing Protocol