qNPA™ Technology
The quantitative nuclease protection assay (qNPA) allows researchers to quickly and accurately measure the gene expression levels in a wide range of mammalian, plant and bacterial cell types. The fast, automated protocol requires no RNA extraction, no RNA amplification, and no RNA labeling, leading to more reproducible results.
Samples are treated in a 96 well plate with our proprietary lysis buffer. Total RNA is available for analysis by qNPA. This fast and simple step removes the need for costly and complicated RNA extraction. Our Lysis Buffer is compatible with many different sample types, including FFPE and primary tissue. For a current list of sample types, click here.
Gene-specific DNA oligonucleotides are added directly to the Lysis Buffer and hybridize to the RNA present in solution. The DNA oligonucleotides are added in excess to ensure that every molecule of RNA capable of hybridizing to an oligonucleotide does so.
S1 nuclease is added to the hybridized sample buffer. The S1 nuclease is a powerful, single-strand specific nuclease which degrades any non-hybridized (non-double-stranded) nucleic acid. This step effectively removes the non-hybridized portion of the targeted RNA (left), all of the non-targeted RNA, and excess DNA oligonucleotides.
The S1 nuclease enzyme is completely inactivated. The RNA::DNA heteroduplexes are then treated to remove the RNA portion of the duplex, leaving only the previously protected oligonucleotide probes.
The resulting DNA oligos are a stoichiometrically representative library of the original RNA sample. The individual DNA probe oligonucleotides are present in the precise relative abundance as the RNA transcripts were in the original sample. The qNPA oligonucleotide library is now ready to be quantified using the ArrayPlate™ Detection System.
