Frequently Asked Questions
Q: What is the significance of the 260/230 ratio?
A: A low 260/230 ratio may indicate contamination of organic or carbohydrate origin. Nucleic acids only absorb light that has a wavelength of 260 nm. Organic contaminants like phenol and other aromatic compounds, TRIzol, and some reagents used in RNA extraction absorb light of a 230 nm wavelength. Samples with a low 260/230 (below about 1.8) have a significant presence of these organic contaminants that may interfere with other downstream processes like RT-PCR or the IVT in Affymetrix® experiments, lowering their efficiency. In order to foster the success of the microarray and gene expression experiments performed at the Microarray Core Facility, we strongly recommend limiting microarray experiments to those samples with a 260/230 ratio greater than 1.8. Running samples with 260/230 ratios lower than 1.8 could have reduced yield and substantially less optimal results. DGML users who wish to run samples that have organic contamination indicated by a low 260/230 ratio must assume responsibility for the performance of their samples. The DGML requires that in order to run samples with low 260/230 ratios, the user must sign off on the samples showing that he/she is aware of the risk associated with performing microarray experiments with samples that do not the meet sample purity standards delineated by the DGML.
Q: What is the best protocol to extract RNA from tissues?
A: There is not a 'best' protocol to isolate and extract RNA from tissues. RNA and DNA tissue will store in RNALater, Qiagen web site. indefinitely.We highly recommend the use of TriZol or Tri-reagents for a better yield (especially if you want to look at micro RNAs- RNeasy kits will remove the smaller RNAs), but we have found that the RNeasy Kits from Qiagen produce very good results. Genomic DNA contamination of RNA samples can cause an increase in false positives. The protocol is also available at the Qiagen web site.
Note: Users who are using very small amounts of tissue and/or cells can use the RNeasy Micro Kit from Qiagen.
Q: Should I use total RNA or polyA RNA for my reactions?
A: We suggest using total RNA for all of our experiments.
Q: What is the minimum requirement for RNA quantity?
A: It would depend on the platform you choose.
Note: Users wishing to use aRNA (amplified RNA), should READ and UNDERSTAND the protocol that they plan to use and that they have VERY GOOD experimental design in order to end up with usable material for submission (i.e. labeled appropriately). Users who wish to do amplification of small quantities of RNA should meet with DGML before starting their experiment to make sure their experimental design is appropriate.
Q: Do I need to bring the spectrophotometer info or gel image for my RNA samples?
A: Yes if you have this information. You can use this information to help decide what dilution factor to provide us with for the initial submission, but we do not use the gel/spec information to decide quality of RNA for microarray experiments. We use the Agilent 2100 Bioanalyzer and the Nanodrop ND-1000 Spectrophotometer as part of our service.
Q: Can I use amplified RNA (aRNA) for my experiments?
A: Yes, where aRNA can be used when your RNA quantity available is too low. The DGML also does provide this service. Please see our price list
Note: Please contact us prior to starting your experiment if you plan to use aRNA for any single-stranded probe hybridization
Q: What do I need to do when I am dropping off samples for full service and/or quality assurance?
A: When dropping off any samples to the DGML, an order request should already have been submitted to us and the user should receive a confirmatory email instructing them to bring the samples for that request to the laboratory. Each user must bring 5.0-6.0 µL of the diluted RNA samples they wish to run at a concentration range of 25-500 ng/µL, or 200-5000 pg/µL for small samples. The bioanalyzer's range of detection for RNA is 25-500 ng for the Nano LabChip or 200-5000 pg for the Pico LabChip, and we need 2.0 µL to run the assay. We also run 1.5µL your sample on the NanoDrop® ND-1000 Spectrophotometer to get an accurate concentration of your sample. We suggest you bring at least 5.0-6.0 µL per sample for error and in case multiple runs are needed.
Genomic DNA samples for Array-CGH should first be tested using the Nanodrop ND-1000 Spectrophotometer and run on a 2% agarose gel to test for integrity.
Q: What do I need to bring for the Affymetrix hybridization service?
A: Some users request only hybridization, staining/washing, or scanning services with the DGML. The samples to be hybridized should first be submitted as an order on our website with information in the notes section to alert the technicians that this sample will by labeled by the user and only hybridized, stained, and scanned in the DGML. Each sample submitted for Affymetrix basic service should be biotinylated cRNA. The DGML will quantify the sample for concentration and fragment the cRNA. We will then prepare the sample(s) for hybridization, staining, washing, and scan the GeneChip®. If the user intends to provide their own GeneChips® they need to bring them to the DGML as well.
Q: How many replicate arrays do I need to do per sample?
A: The DGML strongly recommends at least 4 biological replicates per experimental condition.
Please note: The same sample on different arrays is considered a technical replicate, but the same treatment on different arrays is a biological replicate. Remember, the more replicates you have, the more accurate, reliable, and believable your data will be.