In general, tissue samples should be processed immediately. If processing of the tissue samples is delayed for several hours, the quality of the isolated gDNA will be lower, particularly for metabolically active organ tissues. In many cases, tissue samples need to be stabilized before genomic DNA purification can be performed. Adequate sample storage can be carried out in one of the following ways:
- Flash frozen tissue samples are stored as whole pieces at -80°C.
- Flash frozen tissue samples are pulverized under liquid nitrogen and subsequently stored at -80°C as tissue powder.
- Tissue samples are incubated with stabilizing agents like RNAlater (Thermo Fisher Scientific) to enable transport at room temperature or on ice, or to enable safe mid-term storage at 4°C or -20°C. Additionally, cutting and preparing aliquots of stabilized samples is significantly more convenient than using fresh or frozen samples.
Below is a list of recommendations for preparing tissue samples from each of the 3 options mentioned above.
Fresh and Frozen Tissue Pieces
- Keep fresh samples on ice and frozen samples frozen (e.g. by storing on dry ice). Label and pre-cool reaction tubes on ice or a cooling block.
- Do not use more tissue than recommended (See “Choosing Input Amounts”).
- Cut appropriately-sized tissue fragment into small pieces and weigh out the exact amount by transferring small tissue pieces into reaction tube positioned on a micro balance.
- Keep tubes cold and start lysis as soon as possible.
- Use a clean, frozen cooling block or the bottom side of a frozen metal reaction tube stand for cutting frozen tissue into smallest possible pieces. Samples are most easily cut when they are processed shortly before thawing.
- Weigh the desired amount by transferring small tissue pieces into a pre-chilled reaction tube positioned on a micro balance.
- Keep tubes frozen or on ice, and start lysis as soon as possible. In samples that have been frozen, ice crystals have destroyed cell structures and nucleases have free access to the genomic DNA. Work with the smallest possible tissue pieces to allow for a rapid inactivation of nucleases by Proteinase K. Make sure all tissue pieces are able to move freely in the lysis buffer before immediately starting lysis at 56°C.
Frozen Tissue Powder
- Label and pre-cool reaction tubes on dry ice. Keep tubes containing tissue powder on dry-ice and use small pre-chilled scoops that allow for the transfer of 5 or 10 mg frozen tissue powder at a time. Tare pre-chilled tube on the micro-balance and transfer appropriate amount of frozen tissue powder to tube for weighing. Work quickly to prevent the tube from warming up on the balance. Keep the aliquoted samples on dry ice to ensure the powder stays frozen.
- When adding Proteinase K and Tissue Lysis Buffer, mix immediately so that the tissue powder is released from the tube wall and dispersed evenly over the lysis buffer. It is important to start lysis at 56°C immediately; add the reaction components to one tube, mix and place at 56°C immediately, then proceed with the next tube. Do not dispense Proteinase K and Tissue Lysis Buffer to all tubes at once.
Stabilized Tissue Samples
If stabilized sample was frozen, thaw first. Remove stabilizing solution from the outside of the tissue sample by blotting on a paper towel or other absorbent paper. Cut the tissue sample into small pieces and weigh the desired amount in a reaction tube (see “Choosing Input Amounts”). Keep tubes cold. Although rapid processing of the samples is recommended, it is not as critical as for fresh or frozen samples because of the presence of the stabilizing agent. Stabilized tissues contain proteins that have an altered fiber structure. These proteins are more difficult for Proteinase K to digest and a fraction of insoluble fiber will remain even if lysis is complete and the lysate looks mostly clear. Since these fibers will block the membrane binding sites when the lysate is spun through, centrifugation of the lysate before loading on the column is recommended for best yield and purity. This is particularly important for brain and fibrous tissue samples (e.g. muscle).
- Monarch Genomic DNA Purification Kit Product Manual
- Choosing Input Amounts for the Monarch Genomic DNA Purification Kit
- Troubleshooting Guide for Genomic DNA Extraction & Purification
- Factors Affecting DNA Quality when Purifying gDNA from Blood and Tissues with the Monarch Genomic DNA Purification Kit