Q: What is the significance of the 260/230 ratio in SAQC?
A: A low 260/230 ratio may indicate contamination of organic or carbohydrate origin. For more information see The Significance of the 260/230 Ratio.
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, but we have found that the RNeasy Kits from Qiagen produce very good results. Although it is not necessary, we also strongly suggest using the optional On-Column DNase Digestion found in Appendix D of the complete protocol. Genomic DNA contamination of RNA samples can cause an increase in false positives. The protocol is also available at the Qiagen website.
Note: Users who have very small amounts of tissue and/or cells can use the RNeasy Micro Kit from Qiagen.
Q: What is the minimum requirement for RNA quantity?
A: RNA input requirements are dependent on the application. We are able to process samples starting with as little as picograms of total RNA.
Note: If you require amplification of low amounts of RNA, please set up a meeting with GARP personnel prior to starting your experiment.
Q: Do I need to bring the spectrophotometer info or gel image for my RNA samples?
A: No, GARP does not need this information. Users can use this information to help decide what dilution factor to provide us with for the initial Agilent submission, but we do not use the gel/spec info to decide quality of RNA for downstream applications. We use the Agilent 2100 Bioanalyzer, NanoDrop Spectrophotometer and Qubit Fluorometer as part of our SAQC service.
Q: What do I need to do when I am dropping off samples for full service and/or quality assurance?
A: Prior to dropping off samples to GARP, please enter an SAQC request in GSLE. A confirmation email will be sent to the requester with instructions to bring samples to the core. We request 5.0-6.0 µL of each nucleic acid sample for quality assessment. RNA samples need to be diluted to 25-500ng/uL, or <5ng/uL for low concentration samples. DNA samples need to be diluted to <50ng/uL in order to be run on the Agilent Bioanalyzer.
Please bring all samples in 1.5 ml tubes and put sample ID numbers on the top of the corresponding tubes. (We will not accept 0.6 ml or 0.2 ml tubes.)
Note: After GARP has judged the quality of the material, results of the completed assay will be accessible to the user via GSLE. The information should be used to prepare the RNA or DNA sample. In order to open a result file emailed to you by the GARP (also accessible via GSLE), you will need to download and install the Agilent Expert Review Software for Molecular Assays (DNA/RNA), available for free from the Agilent website.