不同取样和提取方法对甲状腺结节细针穿刺标本提取RNA检测目的基因的影响
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江苏省“兴卫工程”医学重点人才项目(RC2011068)


Effects of different sample collection methods and RNA extraction methods from thyroid fine needle aspiration samples on target gene detection
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    摘要:

    目的:比较不同取样和提取方法对甲状腺结节细针穿刺(fine needle aspiration,FNA)标本提取RNA检测目的基因结果的影响-方法:以单独穿刺与穿刺针冲洗液2种方式收取FNA标本,以TRIzol法和Qiagen法2种方法提取RNA,利用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测甲状腺细胞相关基因,比较不同方法对RNA质与量及RT-PCR结果的影响-结果:102例FNA标本均提取了RNA,RT-PCR结果显示,GAPDH的检出率达97.06%,TG为93.14%,其余依次为TPO-TSHr-NIS-在单独穿刺样本中,TRIzol法提取的RNA浓度及总量均大于Qiagen法,但差异未达统计学意义 (P > 0.05)-2种方法所提取RNA的吸光度值及PCR成功率也无统计学差异 (P > 0.05)-在冲洗液标本中亦未发现Qiagen法与TRIzol法在RNA浓度-总量-吸光度值及PCR成功率之间有显著差异(P > 0.05)-在TRIzol组中,单独穿刺与穿刺针冲洗液2种取样方式对其RNA浓度-RNA总量-吸光度值以及PCR成功率的影响均无统计学意义(P > 0.05)-在Qiagen组中,不同取样方式对其RNA浓度-RNA总量-OD值以及PCR成功率的影响亦无统计学意义 (P > 0.05)-结论:FNA标本可以成功提取RNA并进行基因检测-单独穿刺-穿刺针冲洗液2种取样方式以及TRIzol法-Qiagen法2种RNA提取方法均可用于FNA标本RNA的提取-

    Abstract:

    Objective:To compare the impacts of different sample collection methods and RNA extraction methods from thyroid fine needle aspiration samples on the results of target gene detection. Methods:Trizol and RNeasy Plus Micro Kit (Qiagen) were used to extract RNA from thyroid FNA samples obtained from a separate puncture or leftover material in the needle; the thyrocyte-relevant genes were detected by reverse transcription-polymerase chain reaction; and the influence of different sample types and RNA extraction methods on RNA quality,quantity and RT-PCR outcome was analyzed. Results:RNA was isolated from 102 FNA samples,the RT-PCR results showed that the positive detection rate of GAPDH and TG were 97.06% and 93.14% respectively,followed by TPO,TSHr,and NIS. The concentration and quantity of RNA extracted by Trizol were higher than that by RNeasy Plus Micro Kit in samples from a separate puncture,but the differences were not statistically significant(P > 0.05). The OD ratio of RNA extracted by the two methods had no significant difference,so as the success rate of RT-PCR(P > 0.05). In samples obtained from leftover material in the needle,no significant differences were found in RNA concentration,quantity,OD ratio and success rate of RT-PCR between the two RNA extraction methods (P > 0.05). In the group that RNA was extracted by Trizol,there were no significant correlation between sample collection methods and RNA concentration,quantity,OD ratio or success rate of RT-PCR (P > 0.05). The same results were observed in the group that RNA was extracted by RNeasy Plus Micro Kit,different sample collection methods had no significant impact on RNA concentration,quantity,OD ratio and success rate of RT-PCR (P > 0.05). Conclusion:RNA can be successfully isolated from FNA samples to detect genes. Samples from a separate puncture or leftover material and RNA extraction methods of Trizol or Qiagen can all be used in RNA extraction from thyroid FNA samples.

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张于芝,吕颖钺,陈欢欢,崔 岱,刘晓云,王知笑,段 宇,杨 涛,武晓泓.不同取样和提取方法对甲状腺结节细针穿刺标本提取RNA检测目的基因的影响[J].南京医科大学学报(自然科学版),2014,(12):1667-1670

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  • 收稿日期:2014-06-07
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  • 在线发布日期: 2014-12-26
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