Specimen Collection, Handling, and Nucleic Acid Extraction
Collection and transport
Choice of tube matters. EDTA (lavender) is the preferred anticoagulant for molecular testing; heparin inhibits Taq polymerase and reverse transcriptase, and citrate dilutes plasma and chelates magnesium. Plasma for cell-free DNA should be separated promptly or collected in a stabilizing tube. Swabs should be flocked nylon in appropriate transport medium; some molecular assays accept dry swabs. Tissue should be fresh-frozen or fixed in buffered formalin with controlled time, because prolonged fixation crosslinks and fragments nucleic acids. RNA requires rapid stabilization (chaotropic lysis or RNAlater) and cold chain. Freeze-thaw cycles degrade nucleic acid and cause hemolysis, so aliquot.
Extraction chemistry
Silica-based columns or magnetic beads with chaotropic salts bind nucleic acid; phenol-chloroform separates phases (aqueous on top, protein at the interphase); ethanol or isopropanol precipitates. Proteinase K digests protein; DNase I must be used when pure RNA is required.
Quality assessment
A260 of 1.0 equals 50 µg/mL double-stranded DNA, 40 µg/mL RNA, and 33 µg/mL single-stranded DNA. A260/280 near 1.8 indicates pure DNA and 2.0 pure RNA; A260/230 below about 1.8 suggests chaotropic salts or solvents. Fluorometric methods (Qubit) are more specific than UV absorbance when contaminants are present. RNA integrity is scored by RIN.
Traps
Common wrong answers involve heparin tubes, over-fixed tissue, and believing a high A260 guarantees amplifiable template. Inhibitors include heparin, hemoglobin, melanin, humic acid, SDS, and excess EDTA or calcium.
Worked example
A 1:20 dilution of a DNA sample reads A260 = 0.15, so 0.15 × 50 × 20 = 150 µg/mL.