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Jump To: [Microarray FAQ] [RNA Assessment FAQ] [Agilent Bioanalyzer FAQ]
The Core will extract data from individual chips for you. However NO masking of any type will be done. You will get DAT (image), CHP (data in Affymetrix software format), CEL (averaged intensity), TXT (data in text format, tab limited, same information as CHP) and EXP (general experiment info) files from us. Currently, this absolute analysis is done with MAS v. 5; the Core will not extract data using other software.
The Core provides users with the Affymetrix Suite Analysis software package for limited use. Affymetrix has other packages that are more sophisticated and are available including DMT and Micro DB. Users have access to these programs at the facility and can analyze their data with these software packages; however due to staff shortage we are not providing support for these software packages. Other analysis programs such as GeneSpring are available for data analysis at the Bioinformatics core. Statistical analysis consulting services are available on a charge back system from Biostatistics core. Please check with us for details
Affymetrix replaces defective chips free of charge. Once we have identified a defective chip we will notify you and it will be up to you to seek the replacement of a defective chip. Replacement of a chip can take up to a month. Contact rosemary_obrien@affymetrix.com to arrange replacement. The FedEx address to send CDs with the defective chip data is 3371 Mercer Lane, San Diego, CA 92122. Typically, they will request a copy of the DAT and EXP files for the defective chip. Occasionally, they have requested the chip itself. Please make sure to obtain your defective chips from the facility.
We will create a picture gallery for you to see what type of defects can be encountered and will always inform you when we suspect that your chip is defective.
Affymetrix will hold User Seminars—watch for announcements. The seminars will address uses of MAS and NetAffx applications.
You may request protocols directly from Affymetrix. Our website has protocols available that you can use; these are the same protocols our facility and others on campus use.
If the quality of your RNA appears to be less than optimal you will be contacted and asked to make a decision as to whether to continue with the processing of the particular sample and/or batch of samples. If you decide not to continue processing of faulty sample or wish to provide a replacement, an extra $60 charge will apply.
We typically get very good results with 10 ug of total RNA as starting material. If you decide to use lower or higher amounts you should keep that consistent among all of your experiments if you plan on comparing chips from different experiments. Obviously, the final yield is highly dependent on the quality and cleanliness of the RNA sample. Please be aware that we do not encourage you to use less than 10 ug of starting material. If you decide to start your probe synthesis with less than 10 ug we take NO responsibility for poor results. We will not manipulate the sample in order to attain a desirable working concentration (speed-vacuum/precipitation).
Samples are handled on a first come first served basis. Usually, if you bring in fragmented cRNA labeled probe it will take approximately one week of processing time.
We recommend 15ug; it is your decision. We cannot guarantee optimum results if you use less cRNA. If you decide to use lower or higher amounts you should keep that consistent among all of your experiments if you plan on comparing chips from different experiments.
One possibility is that when you are setting up your IVT reaction you are not letting the components warm up to room temperature. You should let ALL the components of the reaction (including cDNA and with the exception of the enzyme) warm up to room temperature. The reaction buffer contains spermidine, which can precipitate cDNA in cold solutions. Please note that even if your RNA appears to be of good quality impurities in your sample may prevent efficient 1st and 2nd strand cDNA synthesis, this will also result in low IVT yields. The Agilent Bioanalyzer quality check does NOT detect impurities in your RNA, only RNA quality/integrity is examined in this analysis. It is for this reason that the Core recommends isolating RNA using Trizol and then following the extraction with a clean-up procedure using the RNAeasy columns.
You need to bring your samples in the concentration range of 50ng/ul to 500ng/ul and at a volume of at least 2.5ul.
We require that you bring total RNA for analysis.
Results will be available within 2-3 days.
You will be given a printout containing the 28S/18S rRNA ratio along with the RNA concentration in ng/ul and a print out of the electropherogram.
If your computer has the Agilent 2100 Bioanalyzer software we can e-mail you the results. Micorarray Core can install the Agilent 2100 Bioanalyzer software on your computer. Please inquire about the charges. The Agilent 2100 Bioanalyzer files are opened using this software.
The 28S/18S ribosomal RNA ratio should be with in the range of 1.8 to 2.3. Ratios deviating from this range are most likely due to degradation and as a result a broadening either of the 18S peak, 28S peak, or both. The 260/280 ratio is the absorbance of nucleic acid (260nm) to protein (280nm) and is a measure of protein contamination, this ratio should fall within a range of 1.9 to 2.0. The Agilent Bioanalyzer does not give the 260/280 ratio.
The peak that migrates just before 24 seconds is the spike control. It is in all samples including the ladder and is used to align each lane to the 6000 RNA ladder lane.
We would refuse to process a very degraded sample. If, upon further discussion with the core staff, the user insists on having the samples processed, the Principal Investigator will have to make the request. The PI will have to inform Dr. Chaudhry by email of his or her decision.