DNA and RNA extraction by sample type: blood, tissue, plant, fecal, viral and plasmid

The chemistry of a silica-based kit is similar from sample to sample. What changes is lysis and what has to be removed. The table sums it up; the sections below cover each sample.

Collect blood in EDTA tubes. Heparin co-purifies with DNA and inhibits PCR, and is difficult to remove. For DNA, most of it comes from white cells: kits either lyse whole blood directly with proteinase K or first remove red cells with an RBC lysis buffer and work from the white-cell pellet or buffy coat.

For RNA, gene expression changes within hours of collection, so stabilise the sample at the time of draw with a stabilisation tube or reagent, and use a kit made for blood RNA.

• Use small pieces, typically 10–25 mg, to avoid overloading the column.
• For DNA, digest overnight or for a few hours with proteinase K at 56 °C until the tissue dissolves.
• For RNA, freeze immediately in liquid nitrogen or place in an RNA stabilisation reagent, then homogenise in a guanidinium lysis buffer.
• Fibrous tissues such as muscle, skin and heart need mechanical homogenisation with beads or a rotor-stator.

Rigid cell walls need grinding to a fine powder under liquid nitrogen. Polysaccharides make extracts viscous and inhibit polymerases, and polyphenols oxidise and bind DNA, turning it brown. Plant kits include additives such as PVP to remove them, and the classic alternative is the CTAB method, with a full recipe in the extraction methods guide.

Fecal samples combine tough Gram-positive bacteria with strong PCR inhibitors: humic substances from diet, bile salts and complex polysaccharides. Effective fecal kits use vigorous bead beating to break bacterial cell walls evenly, so the community you sequence reflects the sample, and a dedicated inhibitor-removal step before binding. Store stool frozen or in a stabilisation buffer from collection to extraction.

Swabs, serum, plasma and culture supernatants carry very little viral RNA. Viral RNA kits use a guanidinium lysis buffer that inactivates RNases and, for many viruses, the virus itself, and add carrier RNA so the small amount of viral RNA binds the silica efficiently.

• Handle samples in a biosafety cabinet until they are in lysis buffer.
• Use RNase-free tubes and filter tips throughout.
• Include an extraction control so failed extractions do not read as negatives.

A plasmid miniprep uses alkaline lysis. Bacteria are resuspended in a buffer with RNase A, lysed with sodium hydroxide and SDS, which denatures both plasmid and genomic DNA, then neutralised with potassium acetate. Small, supercoiled plasmids snap back into shape and stay in solution, while genomic DNA and proteins precipitate. The cleared lysate goes onto a silica column.

• Do not let lysis run beyond about five minutes, or the plasmid can be damaged.
• Invert gently after adding lysis buffer; vortexing shears genomic DNA into fragments that contaminate the prep.
• For low-copy plasmids, start from a larger culture volume.
• For transfecting mammalian cells, use an endotoxin-free kit.

To recover a PCR product or restriction fragment from a gel, cut the band out with as little extra agarose as possible, minimising exposure to UV light, which damages DNA. Dissolve the slice in a binding buffer at about 50 °C and purify on a silica column. Size the band first against a DNA ladder so you cut the right one.

The extraction range includes genomic DNA, RNA and plasmid kits, and dedicated kits for blood, tissue, plant, fecal and viral RNA samples. Tell us the sample, the amount and your downstream method, and we match the kit. For amplification afterwards, see the qPCR and RT-PCR reagents.