Development of a chemiluminescence-based ribonuclease protection assay.
The ribonuclease protection assay (RPA) is a widely used method for the detection and quantification of specific mRNA transcripts in a complex mixture of total RNA or mRNA molecules. While exhibiting many advantages over other RNA detection methods, RPAs are traditionally performed using radiolabeled probes that often require gel purification steps and lengthy exposure times to visualize results. Moreover, these probes can only be used for 1-2 weeks because of their short isotopic half-life and radiolysis. We report a method that improves the traditional RPA by replacing radiolabeled probes with biotinylated probes and lengthy exposure times with quick, streptavidin/HRP-based chemiluminescent detection technology. Biotinylated probes can be used without get purification and are stable for years, as opposed to weeks. Most importantly, our streptavidin/HRP-based chemiluminescent technology enables us to achieve sensitivity results similar to radioactive RPAs and to detect multiple transcripts in a single sample more efficiently. Furthermore, this new protocol addresses and eliminates the one major drawback unique to using biotinylated probes in chemiluminescent RPAs: a confounding artifact, not seen when running radioactive RPAs but commonly detected when using certain biotinylated rare message probes.
Rosenau
Christopher
C
christopher.rosenau@piercenet.com
Kaboord
Barbara
B
Qoronfleh
M Walid
MW
D000199:Actins / Q000235:genetics; D016477:Artifacts; D019921:Biotinylation; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D016259:Genes, myc; D006735:Horseradish Peroxidase; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009994:Osmolar Concentration; D010761:Phosphorus Radioisotopes / Q000032:analysis; D015347:RNA Probes / Q000737:chemistry; D012330:RNA, Double-Stranded / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D012588:Scintillation Counting; D012680:Sensitivity and Specificity; D019809:Streptavidin
D000199:Actins; D008567:Membranes, Artificial; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D019809:Streptavidin; D006735:Horseradish Peroxidase; D012260:Ribonucleases