The following protocols are recommended for collecting and preparing biosamples for DNA extraction by us. The planned gDNA amount is about 5 ~ 30 ug, which is sufficient for MeDIP/hMeDIP-microarray, sequencing, or qPCR. If you use other protocols or methods, please provide us the details prior your sample submission. You may also use these protocols for your own DNA extraction and submit the DNA samples for the project to us.

Biosafety

If your biosamples were potentially exposed to or contain any biohazardous agents, you must disclose before placing your order and shipping your samples. We will evaluate and determine the biosafety compliance and acceptance of the samples.

DNA Extraction from Cells

Cell Amount Requirement

The recommended cell number per sample is 2x10⁶ cells, which should be sufficient to produce 6 ~ 8 ug gDNA.

Cell culture contaminations, such as mycoplasma, chlamydia bacteria, or fungi, are not controlled by antibacterial antibiotics. Their presence can reach unknowingly high levels to impact your data. Our sample QC does not include any contamination detection.

A. Suspension cells
    1) Harvest cells by centrifugation.
    2) Briefly wash the cells in 2 ~ 5 mL 1X PBS, pellet the cells at low speed, and remove the supernatant.
    3) Store the cell pellet at -80°C. Ship on dry ice.
B. Adherent cells
    1) Aspirate away the culture medium.
    2) Wash the cell monolayer with 2 ~ 5 mL 1X PBS briefly. Discard the 1X PBS.
    3) Trypsinize the cells to help dissociating the adherent cells. Stop the trypsinization and suspend the cells in 5 ~ 10 mL culture medium supplemented with 10% calf bovine serum.
    4) Pellet the cells at low speed and discard the supernatant.
    5) Wash cells with 2 ~ 5mL 1X PBS briefly, pellet the cells at low speed, and discard supernatant.
    6) Store the cell pellet at -80°C. Ship on dry ice.

DNA Extraction from Tissues

Tissue Amount Requirement

The recommended tissue amount per sample is 10 ~ 25 mg tissue mass, which should be sufficient to produce 5 ~ 30 ug gDNA.

The recommended FFPE amount per sample for DNA extraction is 5 ~ 20 sections at 5~10 micron thickness or a paraffin block, which should be sufficient to produce micrograms of gDNA.

A. Fresh frozen in liquid nitrogen
    1) Collect fresh tissue immediately after surgical removal.
    2) Wash the fresh tissue in 1X PBS, spin down the tissues at low speed, and discard supernatant. Add 20% glycerol or 20% DMSO.
    3) Store at -80°C or in liquid nitrogen. Ship on dry ice.
B. FFPE samples
    We do not offer (h)MeDIP-sequencing for FFPE-isolated DNA, because FFPE-isolated DNA, typically expected to be at least partially single-stranded, is not compatible with our MeDIP-sequencing protocol. The quality of DNA isolated from FFPE samples will depend on the technique used to preserve the sample and on other factors such as the age of the block. Below is a fixation protocol:

    1) Fixate tissue sample in 4 ~ 10% formalin immediately after surgical removal.
    2) Use a fixation time of 14 ~ 24 hours.
    3) Completely dehydrate the samples prior to embedding in paraffin.
    4) Store and ship at room temperature.

DNA Extraction from Blood Samples

Blood Amount Requirement

The recommended blood volume per sample is 2 mL for whole blood DNA extraction, or 3 ~ 5 mL for isolated white blood cell DNA extraction.

A. Whole blood collection
    1) Use EDTA or citrate based anticoagulant tubes to collect whole blood. Heparin inhibits downstream enzymatic reactions and should be avoided.
    2) Transfer the blood into ~ 300 uL aliquots.
    3) For short time storage, -20 ~ -80°C is OK. For long term storage, use liquid nitrogen. Ship on dry ice.
B. White blood cells from whole blood
    1) Collect lymphocytes or buffy-coat cells separated from whole blood using a protocol in your laboratory.
    2) Store at -80°C or in liquid nitrogen. Ship on dry ice.

Sample Shipment

Please complete, sign, and email us a Project Form prior to sending your samples. The sample package shall be shipped to the address:

ATTN: Samples Receiving (Project#______ )

Arraystar Inc.

9430 Key West Avenue #128

Rockville, MD 20850

USA

Tel: 888-416-6343

Shipping on Dry Ice

• Use screw-cap tubes as the containers; Snap-cap tubes may pop open during shipment. Seal the cap with Parafilm strip.

• Place the sample tubes in a plastic bag;

• Use 10 kg dry ice as refrigerant (sufficient for both domestic and international);

• Include a copy of the signed Project Form in a waterproof bag, separate from your samples;

• Check the carrier for dry ice/international shipping requirements;

• If possible, avoid a drop-off date with coming weekends, holidays, such as Thursday or Friday;

• Obtain the tracking number and notify by email the Arraystar representative who is working with you for the project.

Sample Receipt Confirmation

All shipments and inquiries should reference the Project Number assigned to your project by Arraystar. You will receive notification from us upon receipt of your samples. We'll notify you if there are any discrepancies between your Sample Submission Form and the contents we receive, or if there are any issues with the physical condition of the samples.

Disclaimer for Low Sample Amount/Quality

For the best results, please provide sufficient-amount and high-quality biosamples to us. If the DNA extracted from your biosamples couldn’t meet the amount and quality requirements, we will contact you for you to decide whether to replace, proceed, or drop the sample from the study. With your consent and approval, it is possible for us to proceed with the experiment. In such a case, you understand the data quality and the success rate may decrease progressively with the lowered sample amount or quality. Although usable data may still be obtainable, we cannot predict for certainty nor do we guarantee the quality and success rate of the data outcomes.

9430 Key West Avenue #128 • Rockville, MD 20850

Toll Free: 1-888-416-6343 • Phone: 240-314-0388 • Fax: 240-238-9860

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