DNA can be extracted from whole blood, fresh or frozen tissues, cultured cells and archival tissues. The methodology depends on the source of the tissue.
DNA extraction from FFPE and tumor tissue dissection
- Starting material for DNA purification should be freshly cut sections of FFPE tissue, up to 3 sections, each with a thickness of up to 10 μm and a surface area of up to 250 mm2, can be combined in one preparation.
- Tumor tissue can be dissected if the tumor tissue area is marked using the H&E slide as a template.
DNA extraction from saliva collected in Oragene® containers
- The recommended volume of saliva is 2 ml. The volume of the Oragene® DNA solution in the kit is 2.0 ml. Therefore the total volume of 2 ml of saliva plus the Oragene® DNA solution should be 4.0 ml.
- The full saliva sample should be collected within 30 minutes and the Oragene® DNA kit should be capped immediately. Waiting longer than 30 minutes may decrease the yield and quality of the DNA.
- Mix the Oragene® DNA/saliva sample in the Oragene® DNA vial by inversion.
DNA extraction from blood-related samples
- If possible, collect blood in EDTA tube to reduce DNA degradation. However, other anticoagulants such ACD (citrate) may also be used successfully.
- The amount of DNA isolated from a blood sample is highly dependent on the number of white blood cells present in the sample and the blood storage conditions. On average, 350ug of DNA is isolated from 10ml of fresh blood sample. Samples frozen at -80°C typically exhibit a 15% decrease in DNA yield. For best results, the blood should be stored at 4°C for less than 5 days or be frozen at -80°C on the same day.
DNA extraction from whole blood
- For short term storage, keep the freshly drawn blood samples at 4°C for less than 5 days. For best results, the fresh blood samples should be delivered to the Core as soon as possible after collection.
- For long term storage, blood samples should be stored at -80°C soon after collection.
- The Core facility does not recommend storing blood samples at -20°C for any length of time. Blood storage at this temperature for as little as 3 days results in decreased DNA yield. Storage at this temperature for 2-4 weeks results in half the yield as the same sample stored.
DNA extraction from buffy coats
- The buffy coat and white blood cell pellets can be stored frozen at -80°C prior to processing.
DNA extraction from cell pellets
- Both fresh and frozen cell pellets can be used for extraction.
Sample submission for blood-related, whole blood, buffy coat, and cell pellet
Please submit samples in secure tubes. Samples brought directly to the Core can be placed in the refrigerator located in the laboratory. Please drop off samples directly to a technician at the Core if they need to be stored at -80°C.
DNA, RNA and Protein extraction from fresh frozen tissue or cell pellet
- Solid tissue or cultured cell samples should be processed immediately or else frozen to minimize the activity of endogenous nucleases.
- Slice tissue into pieces no more than 5 mm thick. Cells should be pelleted. Samples must be immediately snap-frozen in liquid nitrogen or in a cryostat.
- If morphologic preservation is not needed, then place the sample in a –80°C freezer indefinitely until DNA or RNA isolation.
Whole Blood Processing for Lymphocytes and Plasma
- Whole blood should be preserved in ACD tubes and EDTA tubes.
- For plasma and whole blood, completely fill the Vacutainer whenever possible to eliminate dilution from the anticoagulant or preservative and immediately mix the blood by gently and thoroughly inverting the tube five to ten times.
DNA Quality Check by Gel Electrophoresis
- DNA should be stored in TE to reduce the chance of degradation. Samples can be analyzed with concentrations as low as 5ng/ul.
- PCR product for successful pyrosequencing will be amplified with primers designed using Pyromark Assay Design software. Biotin modification is required. Sterile PCR preparation is performed in an AirClean 600 PCR workstation (AirClean Systems).
DNA Quantitation by Picogreen Assay
- DNA should be stored in TE or low TE and protected from multiple freeze-thaws as this will cause fragmentation resulting in low Picogreen concentrations.
- Due to the nature of this assay (only double-stranded DNA is detected), Picogreen readings will often result in significantly lower concentrations when compared to spectrophotometric (260/280) readings. The DNA quantitation by Picogreen Assay is necessary for sensitive downstream applications such as GoldenGate genotyping and Next Generation sequencing.
Consultations with Users
- We offer free consultations to our users throughout the sample processing. Prior to the project start, we encourage the users to contact the BER (Contact Person: Dr. Jian Gu, Director) to discuss their research needs. Then the BER staff will provide the user with guidelines on sample collection and preparation. For the user who has special needs for sample processing, after consultation we will develop a modified service tailored to their needs if possible. In addition, an overview of the service and an estimated turnaround time are provided before service begins.
|BER Service||2015 Price|
|DNA extraction from blood||$30/blood sample|
|DNA extraction from saliva||$22/saliva sample|
|Simultaneous DNA/RNA/protein extraction||$95/sample|
|RNA from fresh frozen tissue||$50/sample|
|DNA from frozen tissue||$28/sample|
|DNA from FFPE tissue (max 3 slides/sample)||$38/sample|
|# of extra FFPE slides||$0.50/slide|
|Processing of whole blood||$6/sample|
|Picogreen||$152/plate of 88 samples|
|Protein extraction from fresh frozen tissue||$18/sample|