Biological activity of mRNA immobilized on nitrocellulose in NaI.
Biological activity of mRNA immobilized on nitrocellulose in NaI.
复制标题
NaI 中固定在硝化纤维素上的 mRNA 的生物活性。
DOI：
10.1073/pnas.80.21.6523
复制
发表时间：
1983
影响因子：
11.1
通讯作者：
Gillespie,D
中科院分区：
文献类型：
--
作者：
Bresser,J;Hubbell,HR;Gillespie,D
在 12.2 摩尔 NaI 和 25°C 或更低温度下，mRNA 与硝化纤维素结合，而 DNA 和 rRNA 则不然。结合不需要聚 (A) 束和帽。固定化的 RNA 可以被翻译、反转录、与放射性探针杂交或释放以进行进一步操作。 mRNA 有效地从聚丙烯酰胺转移到 NaI 中的硝化纤维素上。将 NaI 固定的 mRNA 固定到硝酸纤维素上不需要烘烤。当溶解在 12.2 摩尔 NaI 中的细胞通过硝酸纤维素过滤时，mRNA 被选择性结合（快速印迹）。利用蛋白酶和去污剂制备细胞以溶解 NaI 的快速印迹系统特别适合定量、快速筛选细胞的特定基因表达。在人类白血病细胞中检测到高度重复的 DNA 序列的表达。
In 12.2 molal NaI and at 25 degrees C or below, mRNA bound to nitrocellulose while DNA and rRNA did not. Neither the poly(A) tract nor the cap were required for binding. The immobilized RNA could be translated, reverse transcribed, hybridized with radioactive probes, or released for further manipulation. mRNA was efficiently transferred from polyacrylamide to nitrocellulose in NaI. Baking was not required to fix NaI-immobilized mRNA to nitrocellulose. When cells dissolved in 12.2 molal NaI were filtered through nitrocellulose, mRNA became selectively bound (quickblot). The quick-blot system utilizing protease and detergents to prepare cells for NaI solubilization was especially suitable in quantitative, rapid screening of cells for expression of specific genes. Expression of highly repeated DNA sequences was detected in human leukemia cells.