Krueger L J, Andryuk P J, Borigini M J
J Biol Response Mod. 1986 Dec;5(6):539-47.
Using a new method for the direct measurement of the double-stranded RNA (dsRNA) molecule poly(I).poly(C12, U) in plasma, levels of 100 X 10(-9) g of drug were routinely quantified. The samples were digested by proteinase K in a buffered solution containing 0.1% of Brij-35 and deoxycholate detergents. The digestions were terminated after 1 h by the addition of Brij-58 and boiling saturated NaI (1.67 g/ml). Serially diluted samples were filtered onto nitrocellulose and the filters washed and hybridized. Levels of the hybridized-radioactive probe, synthesized de novo in an RNA dependent DNA transcription system, were determined by liquid scintillation spectrophotometry and quantified by comparison to a standard curve. The efficiency of hybridization declined when the plasma concentration in the reaction fell below 1.0 mg/ml. Incubation and denaturation temperatures significantly altered the amount of radioactive probe hybridized; results varied in the extent of hybridization and in the concentration range of dsRNA showing a linear response. Elevated temperature during proteinase K digestion showed reduced hybridization efficiencies: 100% at 25, 80% at 37, 35% at 45, and 25% at 55 degrees C. Incubation at elevated temperatures, prior to the addition of NaI, caused a decline in the amount of radioactivity hybridized, but did not have an effect during hybridization.
采用一种直接测量血浆中双链RNA（dsRNA）分子聚肌苷酸-聚胞苷酸（12:1）[poly(I).poly(C12, U)]的新方法，常规定量检测出药物水平为100×10⁻⁹g。样品在含有0.1% Brij - 35和脱氧胆酸盐去污剂的缓冲溶液中用蛋白酶K消化。1小时后，加入Brij - 58和沸腾的饱和碘化钠（1.67 g/ml）终止消化。将系列稀释的样品过滤到硝酸纤维素膜上，然后对滤膜进行洗涤和杂交。在RNA依赖性DNA转录系统中重新合成的杂交放射性探针水平，通过液体闪烁分光光度法测定，并与标准曲线比较进行定量。当反应中的血浆浓度降至1.0 mg/ml以下时，杂交效率下降。孵育和变性温度显著改变杂交的放射性探针量；杂交程度和显示线性反应的dsRNA浓度范围的结果各不相同。蛋白酶K消化过程中温度升高显示杂交效率降低：25℃时为100%，37℃时为80%，45℃时为35%，55℃时为25%。在加入碘化钠之前于高温下孵育，导致杂交的放射性量下降，但在杂交过程中没有影响。
