- Quantitation of RNA can be performed using direct spectrophotometric measurement (Nanodrop, Trinean), RNA-specific dye-assisted fluorometric measurements (Qubit®, RiboGreen®), or by RT-qPCR. Each method has advantages and disadvantages relating to accuracy, time requirements, equipment requirements, and expense.
- Direct spectrophotometric analysis of samples with a micro-volume spectrophotometer (Nanodrop) is easy, rapid, and appropriate for routine measurements where absolute concentrations are not required. These devices do not perform well on dilute samples (below 20 ng/μl). Additionally, the contribution of other macromolecules to the absorbance spectra is not always appreciated with these devices. Use of a spectrophotometer with content profiling (Trinean DropSense) can be helpful.
- Fluorescent dyes that specifically bind to RNA can provide a more accurate way to determine concentration but require additional effort because of the need to generate standard curves with samples of known concentration. Many kits exist for this approach, and the overall workflows have been optimized for efficiency, providing a reasonable balance between accuracy and effort/cost. NEB routinely utilized these methods during RNA kit development and sample manipulations.
- RT-qPCR remains the gold standard for absolute quantitation of RNA and provides unrivaled limits of detection. Care must be taken to design appropriate primer sets to detect RNA only and appropriate controls must be utilized to ensure amplification is RNA specific and not from residual host DNA. We recommend NEB’s Luna RT-qPCR products.
RNA Purity & Integrity
- Purity of eluted RNA samples can be quickly assessed by reviewing OD ratios collected during routine spectrophotometry. Pure RNA typically has an A260/280 of 1.9–2.1, and an A260/230 of 2.0–2.2. Many factors can influence these values such as the use of a proper reference blank solution, the buffer pH, and contaminants such as protein, buffer salts, ethanol, etc.