Assessing Nucleic Acid Quality and Quantity

The gate

Before an extract goes into an assay, the lab needs to know how much nucleic acid there is and how clean and intact it is. An abundant but contaminated or degraded extract fails as surely as a dilute one ¹. The answer decides whether a specimen goes forward, gets re-extracted, or is rejected.

UV spectrophotometry

Bases absorb UV maximally at 260 nm ². At a 1-cm path, A260 = 1.0 corresponds to about ³:

Concentration = A260 × factor × dilution. A dsDNA sample diluted 1:10 reading A260 = 0.25 is 0.25 × 50 × 10 = 125 ng/µL. Readings are most reliable in the instrument’s linear range, roughly A260 0.1-1.0; micro-volume instruments read undiluted.

Purity ratios ^{3 1}:

A DNA prep reading well above 1.8 for A260/A280 may carry RNA. A low A260/A230 with a normal A260/A280 is the classic signature of chaotropic salt carryover from a silica kit - a cue to repeat the wash.

The limitation: UV measures everything that absorbs at 260 nm - RNA in a DNA prep, free nucleotides, degraded fragments - so it can overstate usable target.

Fluorometry

Fluorometric assays use dyes that fluoresce only when bound to a specific species - double-stranded DNA, RNA, or single-stranded DNA - and read against standards ¹. Free nucleotides, protein, and other nucleic acid types contribute little signal, so fluorometry is more specific and more sensitive than UV, down to low picogram-per-microliter levels.

Use it whenever an assay calls for a defined input mass - NGS library preparation, for example - or when samples are dilute. It’s normal for a fluorometric dsDNA value to come in lower than the UV value for the same extract.

Integrity

Concentration and purity say nothing about whether the molecules are whole.

• Genomic DNA: intact DNA runs as a tight high-molecular-weight band near the wells; degraded DNA smears down the lane.
• Total RNA: dominated by ribosomal RNA. Intact RNA shows sharp 28S and 18S bands, the 28S about twice as intense as the 18S; degradation fades 28S first and adds a low-molecular-weight smear ³.
• Instrument scores: microfluidic electrophoresis assigns an RNA integrity number (RIN) from 1 (degraded) to 10 (intact) ¹. Many expression assays want RIN above about 7. For FFPE RNA, where every sample is fragmented, labs use DV200: the percentage of RNA fragments longer than 200 nucleotides. Similar scores exist for DNA.

Putting it together

The acceptance thresholds come from the assay’s validation and are documented as part of quality assurance (see the lab operations course).

What to take from this

Concentration = A260 × 50 (dsDNA) or 40 (RNA) × dilution. A260/A280 of about 1.8 (DNA) or 2.0 (RNA) rules out protein and phenol; A260/A230 of about 2.0-2.2 rules out guanidinium and similar carryover. Fluorometry measures a specific target and is preferred for defined inputs. Integrity comes from a gel or a score: a tight high-molecular-weight DNA band, sharp 28S:18S at about 2:1, RIN, or DV200 for FFPE RNA.