|How much total RNA is needed for an experiment?
Since sample isolation may place limitations upon the amounts of RNA available we work with clients to select among the labeling methods supported by the lab to give the best yields of material for the arrays. In general the basic need is for 200ng of total RNA at 30-50ng/ul. An additional 3ul of volume placed in a separate tube from the main sample is requested for use in the RNA QC testing performed before we begin full processing. If your amounts of RNA are different from those requested, please contact us to discuss options.
|What is the typical turn around time for a project?
Generally turn around time (TAT) is dependent upon the size of the queue when your samples are received and the number of samples with your submission. If no samples fail QC, the TAT is generally between two (2) and four (4) weeks. Sets of >20 samples may take additional time but this will be reviewed with you in advance.
|What if I have samples that fail QC testing?
If any samples fail QC, you will be informed and allowed an option to proceed with the others or to replace the failed samples. Any additional time needed for you to replace failed samples is not included in our estimate of TAT.
|Do you concentrate RNA that is below the requested minimum?
We do not concentrate RNA but please contact us to discuss the specifics of your need as we may be able to work out other options.
|Are there any precautions I should take in preparation of my RNA?
Be sure to take measures to ensure RNAse free conditions. Make your best efforts to preserve your RNA when isolating from tissues or cells. This may take the form of use of preservative such as RNAlater or flash freezing in LN2 or in the case of blood use of PaxGene tubes. When extracting your RNA if the use of reagents such as TRIZOL is included, the prep must include a column purification (example: RNeasy) as a final step. Without this final column step, labeling reactions may fail or be attenuated due to contaminants that may be present after organic reagent based extractions.
|Sample quality metrics for RNA?
Samples of total RNA should have 260/280 values between 1.75 and 2.0 and meet a RIN score of 7 or better.
|What is a “RIN” score?
RNA Integrity Number (RIN). This number is derived from the Agilent Bioanalyzer testing of RNA. The RIN value ranges between 1 and 10, with 10 being pure non degraded RNA and 1 being completely degraded RNA. This value is calculated from the Agilent analysis by the Agilent “Expert” Software using the entire scan output from the individual RNA sample. This number is used as a quality metric to aid us in assessment of the integrity of individual RNA samples independent of the relative concentrations. We use this number to inform investigators in advance of any concerns about inclusion of a sample or samples in an analysis. Values below 6 are considered failed and not likely to provide useful or reproducible results.
|Why do we recommend biological replicates?
The probability of successfully detecting a differentially expressed gene depends on three main factors. The true magnitude of the differential expression, magnitude of random variation (background noise), and the number of replications. We recommend biological replicates because they increase the statistical power of the analysis, allow for resolution of any outliers, and lend more robustness to interpretations in changes in gene expression detected by the experiment. A minimum of three (3) biological replicates is recommended. We recognize controlling costs is an essential element for investigators as it is for us. However, weighing the informativeness of the data vs. the cost of results should be a prominent factor in deciding on the design of a project. Perhaps fewer conditions with more replicates may be an acceptable strategy to get the most out of a project.
|How is “Hybridization and Scanning service different from “Full Service”?
Samples for hybridization and scan only service are taken in “as is” condition and presumed to be ready to go when received and we are only responsible for the quality of the chip and proper execution of the appropriate hybridization protocol.