肝素酶和氯化锂对抗肝素的RT-PCR抑制作用的试验条件优化

Optimization of test conditions: heparinase and lithium chloride can reduce the inhibition effect of haparin on β-actin amplifications during RT-PCR.

目的探讨肝素酶和氯化锂在对抗肝素抑制逆转录-聚合酶链反应(RT-PCR)作用的试验条件优化及它们各自的优缺点。方法从随机采集20份人临床肝素抗凝全血中提取总RNA并经电泳鉴定后,每份RNA分为未处理组、肝素酶Ⅰ在0.4U、0.5U即时,30min,60min几种条件下的处理组和氯化锂在-20℃下10min、60min,过夜几种条件下的处理组,再逆转录合成cDNA,分别行看家基因β-actin的PCR反应,经1%琼脂糖凝胶电泳观察和比较获得的目的片段。结果从肝素抗凝全血分别抽提的总RNA,经电泳鉴定完整性好,可以进行逆转录-聚合酶联反应。未经处理的RNA经看家基因β-actin的RT-PCR反应后,未能扩增到目的片段;在肝素酶Ⅰ处理组中,仅肝素酶Ⅰ0.5U,并作用60min条件下能扩增出β-actin片段;在氯化锂处理组中,三种时间段作用下,均能扩增出β-actin片段,并且过夜条件下扩增获得的目的片段的量最多;经优化的试验条件的肝素酶Ⅰ和氯化锂作用RNA后,扩增得到的β-actin片段的量基本一致。结论获得肝素酶Ⅰ和氯化锂处理肝素抑制RT-PCR的试验优化条件,并比较了各自的优缺点,为临床相关试验提供一定帮助。

Objective To investigate whether heparinase and lithium chloride can reduce the inhibitory effect of heparin on β - actin amplifications when reverse transcription - polymerase chain reaction （ RT - PCR） was run in heparinized blood samples. Methods After total RNAs were isolated from 20 clinical heparinized blood samples and identified by electrophoresis, each portion of RNA was divided into three parts; One part did not receive any treatment（ group Ⅰ ）, one part was treated with heparinase Ⅰ 0.4U or 0.5U for very short time, 10 minutes or 60 minutes （group Ⅱ ）, the third part was treated with lithium chloride at -20℃ for 10 minutes, 60 minutes or overnight （group Ⅲ ）. RNAs were then reversely transcribed into cDNAs, and housekeeping gene β - actin amplifications were performed by RT - PCR, target fragments obtained were compared on 1% EB - agarose gel electrophoresis. Results Totai RNAs were isolated from 20 clinicai heparinized blood samples, if their integrity identified by electrophoresis was good, RT- PCR was performed. β -actin amplifications were not observed in group Ⅰ ; in group Ⅱ , β -actin amplifications were observed only in sample treated with heprinase Ⅰ 0.5U for 60 minutes ; β - actin amplifications were observed in all the 3 sub- groups of group Ⅲ, with β - actin amplified at the greatest intensity in sample treated overnight. Heparinase and lithium chloride were equally effective in reducing the inhibitory effect of heparin during RT - PCR, β - actin amplification quantity were not significantly different between the two groups. Conclusion We found that heparinase Ⅰ and lithium chloride can reduce inhibitory effect of heparin on β - actin amplifications when RT - PCR was run in heparinized blood samples, we compared their advantages and disadvantages so that test result can be improved.