BRET-based method for detection of specific RNA species.
RNA detection and quantitation is a common necessity in modern molecular biology research. Most methods, however, are complex and/or time-intensive. Presented here is a BRET (bioluminescene resonance energy transfer)-based method that can accomplish the task of RNA identification quickly and easily. By conjugating BRET enzymes to two different oligonucleotides that are complementary to the same target sequence, probes were developed that could detect RNA using a solution-based assay. This assay was optimized for spacer length between the binding sites (found to be 10 nucleotides), and sensitivity was determined to be 1 microg for a specific species of RNA within a mixed population. Specificity of the assay was assessed using in vitro transcribed cRNA and found to be statistically siginificant ( p = 3.11 x 10 (-6), ANOVA, multiple range test). This assay represents a possibility for a less technically demanding, streamlined alternative to canonical RNA assays.
Walls
Zachary F
ZF
Department of Molecular and Medical Pharmacology, Geffen School of Medicine at UCLA, Los Angeles, California, USA.
Gambhir
Sanjiv S
SS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D049410:Luciferases, Renilla / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; D012313:RNA; D049410:Luciferases, Renilla