RNA Quality Control
Every RNA batch is quantified, its integrity assessed, and its purity checked before shipment or library construction.
RNA quality determines VHH library success.
Degraded or contaminated RNA directly compromises the efficiency of reverse transcription and RT-PCR amplification of the VHH repertoire. That is why every extract is systematically qualified before being released, whether intended for an immune library project or shipped as a naive RNA product.
Three complementary controls, on every batch.
Fluorometric quantification
Concentration is measured by fluorometry (Qubit), a method that uses a dye binding specifically to RNA. Unlike spectrophotometry (e.g. Nanodrop), it is not skewed by contaminants absorbing at 260 nm and provides a more reliable estimate of actual RNA concentration.
Integrity assessment
Integrity is assessed by capillary electrophoresis on an Agilent TapeStation system, which generates an electropherogram of the ribosomal profile (18S and 28S peaks) and calculates a RINe score (RNA Integrity Number equivalent) from 1 (fully degraded RNA) to 10 (fully intact RNA).
Purity control
A260/280 and A260/230 absorbance ratios are checked to detect potential protein, phenol or residual chaotropic-salt contamination from the extraction steps. A pure preparation shows ratios between 1.8 and 2.1.
Electropherogram of a high-integrity total RNA sample.
Two sharp peaks corresponding to the 18S and 28S ribosomal RNAs, a flat baseline and little low-molecular-weight noise: the signature of minimally degraded RNA.
Thresholds applied before batch release.
| Parameter | Method | Acceptance threshold |
|---|---|---|
| Concentration | Fluorometric measurement (Qubit) | Depends on volume and intended use |
| Integrity (RINe) | TapeStation | ≥ 7.0 for library construction |
| Purity A260/280 | Spectrophotometry | 1.8 – 2.1 |
| Purity A260/230 | Spectrophotometry | 1.8 – 2.1 |
| Visual profile check | Electropherogram | Distinct 18S / 28S peaks, flat baseline |
A higher RIN means a more representative VHH library.
A RINe score above 7 is generally required for reliable RT-PCR amplification of the VHH repertoire: degraded RNA produces shorter amplicons that are less representative of the true immune repertoire diversity, reducing the number of functional clones recoverable during phage-display screening.