Presence and Characterization of Cell-Free Seminal RNA in Healthy Individuals: Implications for Noninvasive Disease Diagnosis and Gene Expression Studies of the Male Reproductive System
Presence and Characterization of Cell-Free Seminal RNA in Healthy Individuals: Implications for Noninvasive Disease Diagnosis and Gene Expression Studies of the Male Reproductive System
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DOI：
10.1373/clinchem.2009.131128
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发表时间：
2009-11-01
影响因子：
9.3
通讯作者：
Xiong, Chengliang
中科院分区：
文献类型：
--
作者：
Huang, Shiyun;Li, Honggang;Xiong, Chengliang
背景：我们最近检测了无细胞精液RNA (cfsRNA)，并着手研究它在健康个体中的浓度、完整性、稳定性以及它对核糖核酸酶的保护机制。方法：采用靶向ACTB （actin, beta）转录物5′区的反转录实时荧光定量PCR （RT-qPCR）方法对cfsRNA进行定量，通过微毛细管电泳及ACTB和DDX4 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 4]转录物全长扩增分析cfsRNA的完整性，包括测定ACTB和DDX4转录物不同区域的相对量。通过对ACTB和DDX4转录本不同区域的时程分析来测定cfsRNA的稳定性。为了研究cfsRNA是否以复杂的形式受到保护，我们对精浆进行了两种处理方式：通过不同大小的孔过滤和在RNA回收前进行Triton X-100处理。结果：cfsRN浓度变化范围为0.87 ~ 3.64 mg/L[平均（SD）， 1.75 mg/L (0.92 mg/L)]。大多数cfsRNA以部分降解的形式存在，与5‘扩增子相比，中间和3’扩增子的数量较少。虽然DDX4转录本的3‘区在90分钟内完全降解，但ACTB和DDX4转录本的5’区在24小时内保持稳定。通过022 μ m孔过滤，ACTB和DDX4 mRNA浓度分别降低了72%和61%。Triton X-100处理后，几乎所有精子ACTB和DDX4 mRNA均消失。结论：尽管cfsRNA被部分降解，但它代表了不同的转录物种类，并且在健康个体中数量丰富、相当稳定，且与颗粒相关。cfsRNA可能是男性生殖系统和种系表观遗传学的潜在非侵入性生物标志物。(C) 2009年美国临床化学学会
BACKGROUND: We recently detected cell-free seminal RNA (cfsRNA) and set out to stud), its concentration, integrity, stability in healthy individuals, and mechanisms for its protection from ribonucleases.METHODS: We quantified cfsRNA by reverse-transcription quantitative real-time PCR (RT-qPCR) targeting of the 5' region of the ACTB (actin, beta) transcript, cfsRNA integrity was analyzed by microcapillary electrophoresis and by amplification of full-lenght ACTB and DDX4 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 4] transcripts, including measurement of the relative amounts of different regions of ACTB and DDX4 transcripts. Stability of cfsRNA was measured by time-course analysis of different regions of ACTB and DDX4 transcripts. To investigate whether cfsRNA was protected in complexed forms, we processed seminal plasma in 2 ways: filtration through pores of different sizes and Triton X-100 treatment before RNA recovery.RESULTS: cfsRN concentrations varied from 0.87-3.64 mg/L [mean (SD), 1.75 mg/L (0.92 mg/L)]. Most cfsRNA was present in partially degraded forms, with smaller amounts of middle and 3' amplicons compared with 5' amplicons. Although the 3' region of the DDX4 transcript was degraded completely by 90 min the 5' regions of ACTB and DDX4 transcripts were stable up to 24 h. Filtration through 022-mu m pores reduced ACTB and DDX4 mRNA concentrations by 72% and 61%, respectively. Nearly all seminal ACTB and DDX4 mRNA disappeared after Triton X-100 treatment.CONCLUSIONS: Although cfsRNA was partially degraded, it represented diverse transcript species and was abundant, fairly stable, and associated with particles in healthy individuals. cfsRNA may represent a potential noninvasive biomarker of the male reproductive system and of germline epigenetics. (C) 2009 American Association for Clinical Chemistry