生长抑素RNA探针的制备和初步应用

Preparation and Application of Somatostatin RNA Probe

利用含有外源片段生长抑素(SS)-cDNA的质粒pGEM-3进行扩增,采用HindⅢ使质粒线性化,在T7RNA聚合酶的作用下,以尿苷5'[α-32P]三磷酸盐和其它核苷酸合成了同位素标记的ssRNA探针,而以地高辛(DIG)标记的各种核苷酸的混和物合成了DIG标记的ssRNA探针,并分别与从大鼠脑组织中提取的总RNA进行点杂交,以检测探针的有效性,结果这两种探针均有较强的杂交信号,且与总RNA的量呈一定正相关关系,均可用于检测有关样品中SSmRNA的相对含量.

The plasmids of pGEM-3 containing somatostatin (SS)-cDNA segment were cloned. The cloned plasmids were linearized with HindⅢ. Based on template of SS-cDNA segment, T7RNA Polymerase as synthesis enzyme, isotoped marked and digoxin marked ssRNA probes were respectively prepared with 5'-[α-32p]-UTP and other three NTPs, and four digoxin marked combined NTPs, Two probes were both hybridized with total RNA from brain in rat so as to test their role. The results were that two probes presented strong hybridized signals and have positive relation with total RNA contents. They are both used to determine relative content of somato statin RNA in some samples.