一种椎间盘组织总RNA提取的改良方法

目的改进传统常规法在椎间盘组织中总RNA提取,获得高质量的椎间盘组织总RNA。方法健康成年Newsland白兔,完整取出椎间盘组织,随机分RNA传统方法组和酶消化改良组。传统组,按Trizol法提取总RNA;酶消化改良组,加入0.2%Ⅱ型胶原酶,置于细胞培养箱消化数小时,加入含小牛血清的PBS液终止,离心收集细胞。余步骤同传统组。结果传统组,电泳结果未见28S和18S条带,5S条带部分可见;A260/280比值在1.5左右,蛋白多糖污染重。而酶消化组,电泳结果可见清晰的28S、18S和5S条带,且28S条带的亮度约是18S的两倍;A260/280比值在1.8～2.0之间,无蛋白污染,此法提取的RNA,逆转录后可用于扩增β-actin基因。结论酶消化改良法可望成为椎间盘组织总RNA提取的好方法。RNA片断完整,纯度高,无蛋白污染,不影响后续基因表达（RT-PCR）的分析,并且重复性好,可以推广。

An improvement method of extracting total RNA from spinal disc tissue

Objective To improve the method of extracting total RNA from spinal dis tissue. Methods The intact spinal dis tissue were obtained from the Newsland white rabbit, and divided into traditional RNA extraction group and improvemental group treated with enzymatic digestion. The traditional group directly used the Trizol method, while the improvement group pretreated with 0. 2% Ⅱ collagenase to digest for 3 - 4 hours, stopped the digestion by the PBS that contains bovine serum and harvested cells by centrifugation before using Trizol. Results Analysis quality of RNA by electri,the 28S and 18S bands were not seen and 5S band appeared partly in the traditional group, whereas 28S,18S and 5S were clearly seen in the improvement group,furthermore, the quantity of 28S was double or so comparing to 18S. Then,cDNA reverse transcribed from the latter RNA can amplify the beta-actin gene. Additionally, the latter method disappear the pollution of protein or pely. Conclusion The improvement method with enzymatic digestion could be expected as a better method of extracting total RNA from spinal disc tissue.