Roadblock-qPCR: A simple and inexpensive strategy for targeted measurements of mRNA stability
The steadiness of mRNAs is key to figuring out expression degree and dynamics. Nonetheless, present approaches for measuring transcript half-lives (e.g. transcription shutoff) are usually poisonous or technically advanced. Right here we describe an alternate technique for focused measurements of endogenous mRNA stability that’s easy, cheap, and non-toxic. Cells are first metabolically labelled with the nucleoside analog 4-thiouridine (4sU). Extracted mRNA can then be handled with the thiol-reactive compound N-ethylmaleimide.
This compound modifies 4sU nucleotides and sterically interferes with reverse transcription of 4sU-containing transcripts, disrupting their conversion into cDNA. The decay price of non-4sU-containing pre-existing mRNA can then be monitored by quantitative PCR (qPCR). Importantly, this method avoids the biochemical isolation of 4sU-labelled transcripts and/or RNA-seq evaluation required for different metabolic-labelling methods. In abstract, our methodology combines the simplicity of “transcription shutoff” methods with the accuracy of metabolic-labelling methods for measurements of mRNA stability throughout a variety of half-lives.
Aspiculuris tetraptera, as a parasitic pinworm, is most often detected in laboratory mice, and transmission is mediated by the eggs contained within the faeces of contaminated mice. A extremely delicate and quantitative faeces-based diagnostic instrument can be helpful for the early detection of A. tetraptera to inhibit the growth of an infection. On this research, we developed a quantitative assay that reveals excessive sensitivity in detecting A. tetraptera in faeces utilizing PCR methods.
Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of contaminated mice. To quantitatively detect the small quantity of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes have been used for the quantitative PCR assay (qPCR). The mixture of LNA-based DNA elevated detection sensitivity by greater than 100-fold in comparison with utilizing regular oligo DNAs. The copy variety of the A. tetraptera DNA detected was positively associated to the contaminated faeces-derived genomic DNA with a easy linearity regression within the vary of 20 pg to 15 ng of the genomic DNA. To extra conveniently detect an infection utilizing faeces, the LNA-based TaqMan assay was utilized to the crude fraction of the faeces with out DNA purification. An assay utilizing ethanol precipitation of the faeces yielded outcomes according to these of direct microscopic remark.