Reliable RT-qPCR assays demand careful experimental design, extensive quality control, and transparent data analysis. The Thermo Scientific NanoDrop One Microvolume UV-Vis Spectrophotometer is ideally suited to quantify and qualify nucleic acid samples as a quality control step for downstream RT-qPCR assays. The NanoDrop One Spectrophotometer helps prevent failed experiments by providing accurate nucleic acid concentrations and identifying contaminants that may alter experimental results.
An RT-qPCR assay includes two enzymatic steps. In the first step, RNA is converted to cDNA using reverse transcriptase. Normalizing the amount of RNA going into the reaction helps minimize variability in cDNA production, which can be crucial since reverse transcription reaction efficiencies can change with different RNA concentrations.
During the second step, gene-specific primers are used in combination with cDNA template to determine the relative expression compared to various reference genes.
Reference genes are an important part of this reaction because they are used to normalize small differences in cDNA input. If there are large differences in cDNA amount, normalization based on reference genes can become problematic. The MIQE guidelines require that input RNA quantity and purity are reported in publication.
The NanoDrop One Spectrophotometer is built with Acclaro Sample Intelligence technology, which identifies the presence of common contaminants in samples and calculates an approximate concentration of the contaminant. Common molecules found in nucleic acid extraction kits can overestimate analyte concentrations or denature qPCR polymerases. In either case, the qPCR experiment can fail. The Acclaro software provides corrected nucleic acid concentrations and identifies contaminants, to help minimize time spent troubleshooting and managing failed experiments.
SARS-CoV-2, the virus responsible for COVID-19, is an enveloped, non-segmented, single-stranded positive-sense RNA (+ssRNA) virus and a newly identified β-coronavirus. The SARS-CoV-2 viral lifecycle has yet to be resolved, a critical step to studying infection progression.
NanoDrop Spectrophotometers, one of many Thermo Fisher tools for researching SARS-CoV-2 play a vital role in infectious disease research, as quantifying viral RNA/DNA and identifying contaminants are necessary steps to ensuring accurate downstream results.