Min J, Zassenhaus H P
Department of Microbiology, St. Louis Univ. Medical Center, MO 63104.
Biotechniques. 1992 Dec;13(6):870-4.
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.
我们描述了一种对特定的、放射性标记的RNA序列进行定量的方法。该方法将使用生物素化寡核苷酸对RNA进行杂交选择与所选RNA的凝胶电泳分析相结合。我们证明杂交选择方法具有特异性和定量性。它能使特定RNA序列富集至少600倍。通过在选择前对RNA:寡核苷酸杂交体进行RNase T1消化，然后对所选RNA片段进行凝胶电泳分级分离，特异性和灵敏度提高到至少10000倍富集。此外，这种改进使人们能够对RNA转录本的特定区域进行定量，以及在一个实验中监测几种不同的RNA序列。据估计，该方法的灵敏度足够高，能够检测出十万分之一的特定RNA序列。
