Other RNA Services
RNA extraction and quality control of the extracted nucleic acid is crucial for sequencing, microarray analysis, or other genomic technologies. The CGF has established standard operating procedures for extractions to minimize degradation and contamination of precious patient samples. Quality analysis is performed with the Nanodrop ND1000 spectrophotometer, the Qubit Fluorometer, and the Agilent 2100 Bioanalyzer, which has become the industry standard for RNA quality assessment.
The CGF offers the following extraction and quality analysis services:
- RNA extraction via Qiagen RNeasy kits
- miRNA extraction via Qiagen miRNeasy kits
- RNA extraction from FFPET samples via the Qiagen miRNeasy FFPE kit
- RNA quality analysis via the Agilent Bioanalyzer
- Nucleic acid quantitation via the Nanodrop ND1000
- Nucleic acid quantitation via the Qubit Fluorometer
RNA extraction tissue samples may be requested through the Hillman Tissue and Research Pathology Services (TARPS), and cell culture samples may be requested through the CGF (contact Patricia Petrosko).
For researchers who are collecting tissue and cells 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:
- Fresh tissue should be flash frozen in 2.0 mL round bottom tubes or Nunc tubes in LN2 or dry-ice and maintained at -80°C until submitted to the CGF. Cells should be pelleted in 1.5-2.0 mL tubes, all supernatant removed, and then either: 1) flash frozen in LN2 and maintained at -80°C until submitted to the CGF; or 2) placed in RNAlater at 4°C overnight, then briefly spun down and the RNAlater removed, and then stored at -20°C or -80°C indefinitely until submitted to the CGF.
- FFPET samples may be submitted in one of two ways:
- Submit (4) 10 micron scrolls in round bottom 2.0 mL microcentrifuge tubes
- Submit (10) 10 micron blank slides for FFPET extraction. The CGF can perform macro-dissection of the FFPET slides. The researcher must provide an accompanying H&E slide with the area of interest outlined. If this service is requested, we suggest that the researcher talk with a CGF staff member prior to submitting samples to go over the protocol for identifying the area of interest.
Once samples have been extracted, they will be assessed for quality, quantity, and concentration prior to beginning microarray or sequencing assays.
Investigators will be notified if a sample is of insufficient quantity or quality (failed Quality Control parameters) for optimal array performance.
Technology and Equipment
The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells on a single platform. Learn more at the Agilent website.
The Nanodrop (Thermo Scientific) is a micro–Volume UV–Vis spectrophotometer for nucleic acid and protein quantitation that allows for sample volumes as small as 0.5 μL. It offers:
- Direct, easy measurements in less than 5 seconds – just pipette & wipe. No cuvette or dilutions!
- Full spectral output
- Measures DNA, RNA (A260) and Protein (A280) concentrations and sample purity (260/280 ratio)
- Large concentration range (2 ng/μL – 15,000 ng/μL dsDNA) without dilutions
Learn more at the Nanodrop website.
Life Technologies Qubit Fluorometer
The Qubit® 3.0 Fluorometer utilizes specifically designed fluorometric technology using Molecular Probes® dyes. These fluorescent dyes emit signals ONLY when bound to specific target molecules, even in the presence of free nucleotides or degraded nucleic acids. Qubit® fluorometric quantitation provides the most specific and sensitive DNA and RNA quantitation available, even at low concentrations.
Learn more at the Life Technologies website.
Frequently Asked Questions: RNA extraction services
1. What type 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 microdissection samples.
The following is a list of some sample types we have processed in the lab: prostate, breast, renal cell carcinoma, lung, colon, pancreas, gastroesophageal, melanoma, lymphoma, and zebrafish
2. What 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 microRNA extractions, the CGF uses the Qiagen miRNeasy kit. The miRNeasy kit enables co-purification 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 the recommended method.
3. How much RNA will I get from tissues 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 0.2 mg range from 10-30 ug. Please refer to the chart below provided by Qiagen for typical yields.
4. Can I submit extracted RNA for cleanup?
Yes, the CGF routinely performs cleanup on previously extracted RNA samples using the Qiagen RNeasy kits.
5. 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, which are outlined above.