﻿ Frequently Asked Questions | Genomics | Norris Cotton Cancer Center

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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.

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.


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.

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.

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



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.

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.

A: The DGML strongly recommends at least 4 biological replicates per experimental condition.
