Upgrade your RNA quality control with Lunatic
Blog
RNA is the key component for all kinds of therapeutic and analytical applications. However, contaminants show up in RNA preps no matter the RNA source and these contaminants can interfere with anything from gene expression assays to creating RNA-LNPs.
Proteins and genomic DNA often show up in RNA extracted from tissue, cells, bacteria or plasma, because separating the phases during extraction is a tricky job. Improper washing of the RNA can also leave behind reagent residues. Protein and solvent contaminants can inhibit DNases and lower the amount of recovered RNA, while gDNA can interfere with RT-PCR – as a result, there’ll be increased noise in downstream applications like RNA-seq.
Residual DNA templates are a critical process-related impurity of in vitro RNA transcription. When making RNA-LNPs, this DNA can be incorporated into the nanoparticles alongside the RNA and increase oncogenic, infective, or immunomodulatory risks.
Traditional RNA quality control by UV/Vis spectroscopy involves checking if the A260/A280 ratio is ~2.0, indicating pure RNA. However, a ratio below 2.0 says nothing about the identity of the contaminants and it can leave you in the dark about the actual RNA quantity. This is where Lunatic and its Unmix applications flex their muscles.
Handle a broad range of RNA/DNA ratios
Lunatic’s Unmix applications see through contaminants to separate RNA absorbance from co-absorbing impurities. When DNA is the major contaminant in your sample, Lunatic checks RNA quality and quantity for you at the same time. The “RNA (Any source)” app reported negligible amounts of impurities in a sample of pure RNA but detected impurities in a sample with 20% DNA. Lunatic accurately reported the RNA concentration in both cases.
Tackle lots or a little bit of RNA
Samples of purified RNA were mixed with 5% to 40% DNA while maintaining the total nucleic acid concentration at approximately 500 ng/µL. The measured RNA concentrations excellently matched the target values as the proportion of DNA contamination increased.
RNA purification can yield a lot or a little bit of RNA, so a good RNA quality control method needs to cover a wide range of concentrations. RNA and DNA were mixed at a 70/30 ratio to a total nucleic acid concentration of 1,000 ng/µL, then diluted. When checked with Lunatic, the measured RNA of all the dilutions was 70±2% of the total nucleic acid concentration, spot-on the expected value. Unmix gives Lunatic the power to report the actual RNA amount in DNA-contaminated samples across a broad range of nucleic acid concentrations and purities.
Proteins are no problem
Proteins are also common contaminants in RNA extractions so Lunatic’s Unmix deconvolutes nucleic acids from proteins as well. RNA at ~2,000 ng/µL was mixed 9:1 or 1:1 with BSA solution. Lunatic’s Unmix app detected the protein and measured the expected RNA concentration regardless of the contaminant. With Lunatic you don’t have to worry that contaminating proteins will go undetected.
TL;DR
Pure RNA is the critical starting point for many rapidly growing therapeutic and analytical applications, but RNA often suffers from unfavorable contamination even after purification. Lunatic upgrades RNA quality control and can see through contamination from DNA and protein to measure RNA concentration and keep an eye out for impurities. There’s no need to prep dyes, run a standard curve, or risk introducing pipetting errors from sample dilutions. Just load your samples, press start and measure with high-throughput and precision.
