University of Pittsburgh Cancer Institute (UPCI)
Clinical Genomics Facility FAQ
RNA Extraction Service FAQ
What types of samples can I submit for RNA extraction?
The CGF has experience in extracting RNA from a wide array of frozen tissue, cell culture and cell lines from both human and animal subjects, including metatstatic cases. Currently we are working to develop extraction methods for FFPET and laser captured micro dissection samples. The following is a list of some sample types we have processed in the lab: Prostate, Breast, Lung, Colon, Pancreas, Gastroesophageal, Melanoma, Lymphoma, and Zebrafish.
Which RNA extraction method do you use?
The standard and preferred extraction method of the CGF is the Qiagen RNeasy kit. This is a silica- gel- membrane extraction that utilizes a guanidine-isothiocyanate lysis to produce high quality, clean total RNA.
For micro RNA extractions, the CGF uses the Qiagen miRNeasy kit. The miRNeasy kit enables copurification of total RNA including miRNA or enrichment of miRNA in a separate fraction.
At the request of the Investigator, the CGF will perform a standard Trizol RNA extraction, however this is not recommended as the preferred method.
Qiagen Kit Links:
How much RNA will I get from tissue or cells?
The amount of RNA extracted varies and will depend on the sample type, the amount of sample submitted, and measures taken to prevent degradation at the time of collection and storage. Although the lab cannot guarantee RNA quality or quantity due to previously described variables, typical yields for tissue samples of 0.2mg range from 10-30ug.
Can I submit extracted RNA for cleanup?
Yes, the CGF routinely performs cleanup on previously extracted RNA samples using the Qiagen RNeasy kits.
What sample collection methods should I follow to help minimize RNA degradation?
For researchers who are collecting tissue on their own, we suggest a few guidelines to limit RNA degradation and ensure sample quality. All samples submitted to the lab for extraction and downstream microarray applications should be handled in one of the following ways at the time of collection:
- Tissue and cell pellets should be snap frozen in liquid nitrogen and then stored at -80�C until submitted to the CGF.
- Tissue and cell pellets should be placed in RNAlater (Ambion, Inc) and stored at 4�C for up to one month or -20�C indefinitely until submitted to the CGF.
- Tissue and cell pellets should be homogenized immediately in either Qiagen Buffer RLT (recommended- see link below) or Trizol (not recommended) and then stored at -80�C until submitted to the CGF.
For more information on how to limit RNA degradation please see the following Tech Note provided by Ambion, Inc: http://www.ambion.com/techlib/tn/91/9113.html
For product and ordering information for RNAlater: http://www.ambion.com/catalog/ProdGrp.html?fkApp=1&fkProdGrp=51
For ordering information for Qiagen Buffer RLT: http://www1.qiagen.com/Products/Accessories/Buffers/BufferRLT.aspx
