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Assessing RNA (NEB #T2110)

RNA Quantification

  • Quantitation of RNA can be performed using direct spectrophotometric measurement (Nanodrop™, Lunatic), 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), nor do they allow de-convolution of the contribution of genomic DNA to the absorbance at 260 nm. Additionally, the contribution of other macromolecules to the absorbance spectra is not always appreciated with these devices. A spectrophotometer with content profiling (Lunatic UV/Vis 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.

  • RT-qPCR remains the gold standard for absolute RNA quantitation and provides unrivaled detection limits. Care must be taken to design appropriate primer sets to detect RNA only. 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

  • The 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 using a proper reference blank solution, the buffer pH, and contaminants such as protein, buffer salts, ethanol, etc.

  • RNA integrity can be assessed using an Agilent Bioanalyzer, Agilent TapeStation, or a similar device to resolve the sample and determine the ratio of 28S to 18S rRNA, as well as the amount of lower molecular weight species. This approach produces an RNA Integrity Number (RIN for Bioanalyzer, RINe for TapeStation) which encapsulates these generally agreed-upon metrics RIN values greater than 7 are often referred to as "good," though the specific downstream application of the purified RNA determines the true needs of RNA quality. Researchers are encouraged to establish their own criteria.

  • Note that the RNA Integrity metrics from Agilent’s automated electrophoresis systems apply to samples with typical upper and lower ribosomal fragments. For samples with atypical ribosomal profiles such as green tissues of plants with plastidal RNA, and insects with breaks in upper ribosomal fragment, the instrument algorithm is not the best predictor of RNA quality and users may observe a discrepancy between instrument-generated RIN and the actual visual profile of the gel trace. Additionally, there is a growing understanding within the scientific community regarding comparisons between RIN (from Bioanalyzer) and RINe (from TapeStation). Users are encouraged to learn more about the RNA integrity metrics through available resources such as manufacturer's technical notes and peer-reviewed literature that may help set expectations for their sample type.