Quantitation of radioactively labeled RNA by hybrid selection using biotinylated oligonucleotides.
We describe a procedure to quantify specific, radioactively labeled RNA sequences. This procedure combines hybrid selection of an RNA using biotinylated oligonucleotides with gel electrophoretic analysis of the selected RNA. We show that the hybrid selection procedure is specific and quantitative. It enriches a specific RNA sequence at least 600-fold. Specificity and sensitivity are increased to at least 10,000-fold enrichment by a combination of RNase T1 digestion of the RNA:oligonucleotide hybrid prior to selection, followed by gel electrophoretic fractionation of the selected RNA fragment. Furthermore, this modification allows one to quantify specific regions of an RNA transcript, as well as to monitor several different RNA sequences in one experiment. It is estimated that the sensitivity of this procedure is high enough to detect specific RNA sequences present at 1 part in 100,000.
Min
J
J
Department of Microbiology, St. Louis Univ. Medical Center, MO 63104.
Zassenhaus
H P
HP
D001483:Base Sequence; D001710:Biotin; D004587:Electrophoresis, Agar Gel; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D012331:RNA, Fungal / Q000032:analysis* / Q000235:genetics*; D006163:Ribonuclease T1; D012441:Saccharomyces cerevisiae / Q000235:genetics
D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D012331:RNA, Fungal; D001710:Biotin; D006163:Ribonuclease T1