DNA/RNA extraction

Principle

• Isolates nucleic acids from cells while removing proteins, lipids and PCR inhibitors, in four stages: lysis, separation of contaminants, purification, elution.
• Lysis: detergents (e.g. SDS), chaotropic salts (guanidinium thiocyanate or guanidine HCl) and proteinase K break membranes and denature proteins and nucleases.
• Organic method: phenol–chloroform–isoamyl alcohol (25:24:1) partitions proteins into the organic phase; DNA stays in the aqueous phase and is precipitated with ethanol or isopropanol and salt.
• Silica column or magnetic bead methods: nucleic acid binds silica in high chaotropic salt, is washed with ethanol buffers and eluted in low-salt buffer (TE) or nuclease-free water.
• Salting-out methods remove proteins with concentrated salt.

Specimen

• EDTA whole blood (heparin inhibits PCR), tissue, swabs in transport medium, sputum, CSF, plasma, FFPE sections.

RNA-specific points

• RNA is labile; RNases are stable and everywhere. Use RNase-free tubes, tips and gloves, and work on ice.
• Guanidinium thiocyanate–phenol (e.g. TRIzol) with acidic phenol keeps RNA in the aqueous phase while DNA goes to the interphase.
• DNase treatment removes contaminating genomic DNA. Store RNA at –80 °C.

Quality control

• A260 of 1.0 ≈ 50 µg/mL double-stranded DNA, 40 µg/mL RNA, 33 µg/mL single-stranded DNA.
• A260/A280 about 1.8 for pure DNA and about 2.0 for RNA; lower values suggest protein or phenol.
• A260/A230 about 2.0–2.2; low values suggest guanidine salts, phenol or carbohydrate carry-over.
• Integrity: agarose gel (high molecular weight DNA band; 28S and 18S rRNA) or RNA integrity number (RIN).
• Fluorometric methods measure concentration more specifically than absorbance.

Pitfalls

• Include an extraction (negative) control and an internal control to detect contamination and inhibition.
• Over-drying pellets, ethanol carry-over and repeated freeze–thaw reduce yield and quality.

Clinical use

• First step for PCR, qPCR, sequencing, genotyping and molecular diagnosis of infections, cancers and inherited diseases.