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Spotlight on miRNA MicroRNAs (miRNAs)—bits of RNA use Rluc in a hybridization assay. Rluc Rluc–miR21 and a biotinylated form of 18–24 nucleotides in length that can reacts with coelenterazine to emit light anti-miR21 to be sure that the bulbous regulate gene expression—are impliat a maximum of 485 nm. This light Rluc attachment did not hinder the cated in human afflictions such as cancan be detected with a high S/N that miRNA–anti-miRNA interaction. cer, viral infections, and neurological allows detection into the attomole to But how would Rluc–miR21 stack illnesses. An active area of research is zeptomole range. up against unlabeled miR21—the relthe development of miRNA assays with A particular miRNA known as evant cellular species for hybridization which to diagnose disease and monitor miR21 was selected to test the Rluc with anti-miR21? The answer was, fortreatment regimes. In a paper published hybridization assay; miR21 was chosen tunately, that they performed just the in AC (2008, 80, 2319–2325), Sapna because previous studies have shown same. Now, the researchers needed to Deo and co-workers at the calibrate the assay. This was (a) (b) Indiana University–Purdue accomplished by mixing University Indianapolis reknown quantities of Rluc– port on the development of miR21 and anti-miR21 an assay that uses Renilla with varying quantities of luciferase (Rluc) to illumimiR21. As the miR21 con+ + nate miRNA in breast cancer centration was increased, B B cells. more unhybridized Rluc– B Neutravidin plate “Northern blotting is the miR21 was washed away, Biotinylated Rluc– miR21 Bioluminescence gold standard right now” of and the resulting plate’s anti-miR21 miR21 emission current miRNA detection luminosity decreased. The methods, says Deo. “But researchers established a dethese [techniques] are cumSchematic diagram showing the steps in the miRNA hybridization tection limit of 1 fmol, with bersome and not sensitive assay. (a) The anti-miR21, Rluc–miR21 probe, and miR21 mixture is a range of 1 × 10 –11 to 5 × enough.” Other methods (b) immobilized on a neutravidin plate, which is washed prior to coe 10 –15 mol. With replicate for miRNA detection, such lenterazine addition and luminescence detection. data, the assay showed a as microarrays and PCR, variability of
