浮游状态和生物膜状态白念珠菌总RNA提取方法的比较研究

doi:10.13591/j.cnki.kqyx.2022.05.006

口腔医学 ›› 2022, Vol. 42 ›› Issue (5): 416-419.doi: 10.13591/j.cnki.kqyx.2022.05.006

浮游状态和生物膜状态白念珠菌总RNA提取方法的比较研究

段薇, 刘思琪, 黄云, 顾宁, 魏昕

南京医科大学附属口腔医院牙体牙髓科,江苏省口腔疾病研究重点实验室,江苏省口腔转化医学工程研究中心,江苏南京(210029)

修回日期:2022-01-28 发布日期:2022-05-24
通讯作者: 魏昕 Tel:(025)69593035 E-mail:weixinart@163.com
基金资助:
国家自然科学基金(81970945)

A comparative study on the extraction methods of Candida albicans in the form of planktonic and biofilm

DUAN Wei, LIU Siqi, HUANG Yun, GU Ning, WEI Xin

Department of Operative Dentistry and Endodontics, Affiliated Somatological Hospital of Nanjing Medical University; Jiangsu Province Key Laboratory of Oral Diseases; Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing 210029, China

Revised:2022-01-28 Published:2022-05-24

摘要/Abstract

摘要： 目的本研究通过比较不同方法提取生物膜和浮游状态白念珠菌总RNA,以提供一种优化提取白念珠菌总RNA的有效方法。方法将白念珠菌标准株SC5314在生物膜和浮游状态下培养24 h后,分别采用改良Trizol法和改良热酸酶酚法提取白念珠菌总RNA,用紫外分光光度计检测其纯度和浓度,琼脂糖凝胶电泳检测RNA的完整性,通过逆转录PCR反应验证总RNA的完整性及质量。结果与改良热酸酶酚法相比,采用改良Trizol法提取白念珠菌总RNA操作时间较少(P＜0.000 5),提取的总RNA纯度较高(P＜0.05)。琼脂糖凝胶电泳显示两种方法提取的白念珠菌总RNA均呈现28S rRNA、18S rRNA和5S rRNA共3条清晰的条带,逆转录PCR反应也都获得清晰的目的条带。结论改良Trizol法提取白念珠菌成熟期生物膜和浮游状态白念珠菌总RNA的纯度稳定,目前是一种高效、快捷、易于掌握的提取方法。

关键词: 白念珠菌, RNA提取, Trizol, 生物膜, 浮游

Abstract: Objective To provide an effective method to obtain total RNA of Candida albicans(C.albicans) in biofilm and planktonic states by comparing different extraction approaches. Methods Biofilms and planktonic C.albicans SC5314 were incubated for 24 h. Total RNA of C. albicans were extracted using modified Trizol method and modified hot phenol method, respectively. The difference in output, purity and integrity of total RNA extracted by two methods were compared by ultraviolet spectrophotometer, and further confirmed using agarose gel electrophoresis and RT-PCR. Results Modified Trizol method appeared more efficient than modified hot phenol method with less operation time (P＜0.000 5). The purity of the total RNA extracted by modified Trizol method was higher than that by modified hot phenol method (P<0.05). The result of electrophoresis showed that there were three clear bands, 28S rRNA, 18S rRNA and 5S rRNA in total RNA extracted by both modified Trizol method and modified hot phenol method. The result of RT-PCR confirmed the purity and integrity of total RNA of C.albicans with clear target bands. Conclusion Modified Trizol method contributes to more stable purity of the total RNA of C.albicans extracted in the form of planktonic than modified hot phenol method. Moreover, the modified Trizol method is rapid with easy operation and can be applied in experiments with efficiency.

Key words: Candida albicans, RNA extraction, Trizol, biofilm, planktonic

中图分类号:

R379.4

段薇, 刘思琪, 黄云, 顾宁, 魏昕. 浮游状态和生物膜状态白念珠菌总RNA提取方法的比较研究[J]. 口腔医学, 2022, 42(5): 416-419.

DUAN Wei, LIU Siqi, HUANG Yun, GU Ning, WEI Xin. A comparative study on the extraction methods of Candida albicans in the form of planktonic and biofilm[J]. Stomatology, 2022, 42(5): 416-419.

参考文献

[1] Arita GS, Faria DR, Sakita KM,et al. Impact of serial systemic infection on Candida albicans virulence factors[J]. Future Microbiol, 2020, 15: 1249-1263.
[2] 李冉冉, 王兵, 胡胜, 等. 不同RNA提取方法的效能比较[J]. 刑事技术, 2018, 43(6):431-435.
[3] 尚华,彭清,刘佳君,等.类风湿关节炎滑膜成纤维样细胞自噬相关长链非编码RNA筛选与分析[J].中南大学学报(医学版), 2021, 46(10):1071-1079.
[4] Kim Tiam S, Comte K, Dalle C,et al. Development of a new extraction method based on high-intensity ultra-sonication to study RNA regulation of the filamentous cyanobacteria Planktothrix[J]. PLoS One, 2019, 14(9):e0222029.
[5] Poutoglidou F, Saitis A, Pourzitaki C,et al. An improved method for isolating high-quality RNA from rat bone at room temperature without the need for specialized equipment[J]. Cureus, 2021, 13(3):e13806.
[6] 张琰, 马鸣, 虞丽华, 等. 改良热酸酚法提取白色念珠菌早期生物膜总RNA的研究[J]. 口腔生物医学, 2010, 1(2):65-67.
[7] Apip C, Martínez A, Meléndrez M,et al. An in vitro study on the inhibition and ultrastructural alterations of Candida albicans biofilm by zinc oxide nanowires in a PMMA matrix[J]. Saudi Dent J, 2021, 33(8):944-953.
[8] Li YJ, Du JY, Huang S,et al. Hydrogen peroxide potentiates antimicrobial photodynamic therapy in eliminating Candida albicans and Streptococcus mutans dual-species biofilm from denture base[J]. Photodiagnosis Photodyn Ther, 2021, 37: 102691.
[9] Cravener M, Mitchell A. Candida albicans culture, cell harvest-ing, and total RNA extraction[J]. Bio Protoc, 2020, 10(21):e3803.
[10] Jothi R, Hari Prasath N, Gowrishankar S,et al. Bacterial quorum-sensing molecules as promising natural inhibitors of Candida albicans virulence dimorphism: An in silico and in vitro study[J]. Front Cell Infect Microbiol, 2021, 11: 781790.
[11] Kamli MR, Malik MA, Lone SA,et al. Beta vulgaris assisted fabrication of novel Ag-Cu bimetallic nanoparticles for growth inhibition and virulence in Candida albicans[J]. Pharmaceutics, 2021, 13(11):1957.
[12] 梅一堃, 谭镜璁, 王安君, 等. 白念珠菌细胞壁结构及其与宿主相互作用的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(9):1246-1251.
[13] 安佳星, 伍时华, 曾令杰, 等. 不同方法提取酿酒酵母总RNA的比较[J]. 中国酿造, 2021, 40(11):82-86.
[14] 赵仲玖, 廖东升, 邱坤, 等. 利用基于超声原理的动植物组织消融仪快速提取RNA的方法: CN109609500A[P]. 2019-04-12.
[15] 林震嘉, 张辉, 揭英桃,等. 改良的Trizol法提取小鼠皮肤RNA技术[J]. 中山大学学报(医学科学版), 2020, 41(2):224-232.
[16] Greco M, Sáez CA, Brown MT,et al. A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpussiliculosus, the model brown alga[J]. PLoS One, 2014, 9(5):e96470.
[17] 祁文瑾. 新型假丝酵母菌RNA提取试剂及其使用方法: CN103695415A[P]. 2014-04-02.
[18] Kong WJ, Wang Y, Wang Q,et al. Comparison of three methods for isolation of nucleic acids from membranate inner ear tissue of rats[J]. Chin Med J (Engl), 2006, 119(12):986-990.
[19] Coon J, Kingsley K, Howard KM. miR-365 (microRNA):Potential biomarker in oral squamous cell carcinoma exosomes and extracellular vesicles[J]. Int J Mol Sci, 2020,21(15):5317.
[20] Ruocco N, Costantini S, Zupo V,et al. High-quality RNA extraction from the sea urchin Paracentrotuslividus embryos[J]. PLoS One, 2017, 12(2):e0172171.

浮游状态和生物膜状态白念珠菌总RNA提取方法的比较研究

A comparative study on the extraction methods of Candida albicans in the form of planktonic and biofilm

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