Other DNA Services
DNA 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 DNA quality assessment.
The CGF offers the following extraction and quality analysis services:
- DNA extraction via Qiagen DNeasy kits
- DNA extraction from whole blood via Qiagen Flexigene kit
- DNA deparaffinization
- DNA extraction from FFPET samples via the Qiagen QIAamp FFPE tissue kits
- Nucleic acid quantitation via Nanodrop ND1000
- Nucleic acid quantitation via the Qubit Fluorometer
DNA 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 flash frozen in LN2 and maintained at -80°C until submitted to the CGF.
- FFPET samples may be submitted in one of two ways:
- Submit (10) 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.