2种微小RNA实时定量PCR检测方法的比较

目的:对目前最常用的检测微小RNA(miRNA)的茎环实时定量PCR法和PAP实时定量PCR法进行比较。方法:分别用茎环实时定量PCR法和PAP实时定量PCR法检测人乳腺癌细胞MCF-7中U6和23种miRNA的表达,利用定量PCR分析软件和琼脂糖凝胶电泳方法,将2种方法在引物设计难度、特异性与灵敏度,以及检测通量方面进行比较。结果:茎环法的特异性和灵敏度比PAP法高,但引物设计难度大,检测通量低;PAP法引物设计难度较低,检测通量较高,但特异性和灵敏度较差。结论:茎环法实时定量PCR适于有针对性地检测小规模miRNA,而PAP法则适于大规模miRNA筛选实验。     

高糖诱导腹膜间皮细胞发生上皮间质转化中microRNA的异常变化

目的:定腹膜间皮细胞在高糖引起上皮间质转化(Epithelial-mesenchymal transition,EMT)过程中microRNA的表达差异。方法:常规培养PMC细胞,利用高糖培养液刺激诱导腹膜间皮细胞发生EMT,倒置显微镜观察各组细胞形态学变化,实时定量PCR检测EMT标志基因变化,以此确定高糖诱导EMT的发生。利用特异茎环结构的引物合成microRNA的cDNA,实时定量PCR检测重要microRNA的表达变化。结果:高糖培养液培养腹膜间皮细胞48hr后,细胞形态呈梭形改变,同时EMT标记基因E-cadherin表达明显减低(P0.01),Vimentin表达显著升高(P0.01),说明高糖诱导腹膜间皮细胞发生了EMT。利用microRNA特异的茎环结构引物,实时定量PCR检测结果发现高糖刺激后miR-193a的表达明显上调(P0.01);miR-15a和let-7e的表达明显降低(P0.01);miR-16和miR-21的表达无明显变化(P0.05),同时检测发现高糖刺激后miR-193a的表达水平随刺激时间表达升高。结论:异常变化的microRNA可能对高糖诱导的腹膜间皮细胞EMT具有重要调控作用。     

高糖诱导腹膜间皮细胞发生上皮问质转化中microRNA的异常变化

目的：定腹膜间皮细胞在高糖引起上皮间质转化（Epithelial—mesenchymnal transition,EMT）过程中microRNA的表达差异。方法：常规培养PMC细胞,利用高糖培养液刺激诱导腹膜间皮细胞发生EMT,倒置显微镜观察各组细胞形态学变化,实时定量PCR检测EMT标志基因变化,以此确定高糖诱导EMT的发生。利用特异茎环结构的引物合成microRNA的cDNA,实时定量PCR检测重要microRNA的表达变化。结果：高糖培养液培养腹膜间皮细胞48hr后,细胞形态呈梭形改变,同时EMT标记基因E—cadherin表达明显减低（P〈0．01）,Vimentin表达显著升高（P〈0．01）,说明高糖诱导腹膜间皮细胞发生了EMT。利用microRNA特异的茎环结构引物,实时定量PCR检测结果发现高糖刺激后miR-193a的表达明显上调（P〈0．01）;miR-15a和let-7e的表达明显降低（P〈0．01）;miR-16和miR-21的表达无明显变化（P〉0．05）,同时检测发现高糖刺激后miR-193a的表达水平随刺激时间表达升高。结论：异常变化的microRNA可能对高糖诱导的腹膜间皮细胞EMT具有重要调控作用。     

卫氏并殖吸虫PCR和实时荧光PCR快速检测方法的建立

目的建立快速、灵敏、特异的分子生物学检测卫氏并殖吸虫的方法。方法根据卫氏并殖吸虫的特异性基因序列,设计适合于PCR检测的特异性引物及实时荧光PCR特异性引物和探针,并进行灵敏性和特异性试验。结果设计的引物和探针特异性强,所建立的检测方法灵敏度高。应用实时荧光PCR方法的检测灵敏度可达到0.1 pg/μL,比PCR方法的灵敏度高三个数量级。结论本研究所建立的PCR和实时荧光PCR技术检测卫氏并殖吸虫方法的特异性强,灵敏度高,为卫氏并殖吸虫感染的诊治提供了快速的检测技术手段。     

SYBR green实时荧光定量PCR检测微小RNA21的方法建立及在食管鳞癌中的应用

目的:建立SYBR green实时荧光定量PCR检测微小RNA miR-21的技术平台及应用。方法:设计微小RNA21和U6的的颈环结构反转录引物和PCR扩增引物,以U6为内参利用SYBR green实时荧光定量PCR法检测小鼠各器官中的微小RNA21的含量。提取16例食管鳞癌患者的肿瘤组织及其近旁组织中的总RNA,检测其微小RNA21表达水平。结果:SYBR green实时荧光定量PCR检测U6和微小RNA21含量的熔解曲线单一,PCR产物特异。在Balb/c小鼠的4种器官中,肝脏、脾脏、肾脏分别为脑组织的8.71、5.38、3.47倍。16对食管鳞癌患者的样本中,14例微小RNA21的拷贝数高于其近旁组织约10.58倍(p0.01)。结论:此研究成功建立了SYBR green荧光定量PCR法检测小鼠和人微小RNA-21含量的技术平台,为进一步阐述miR-21在食管鳞癌的发生中的作用提供了新方向。     

小菜蛾幼虫偏好表达的microRNA鉴定及功能研究

[目的]microRNA(miRNA)在昆虫生长发育中发挥重要功能,本研究拟通过鉴定小菜蛾不同发育阶段的miRNA,挖掘幼虫偏好表达的miRNA及其潜在功能.[方法]对小菜蛾卵、3龄幼虫、蛹和成虫的miRNA开展高通量测序,结合生物信息学分析方法,筛选在幼虫期偏好表达的miRNA;借助实时荧光定量PCR技术,验证候选m...     

茎环法RT-qPCR定量检测细胞抗猪瘟病毒siRNA的表达

RNAi(RNA interference)已成为特异抗病毒治疗研究的热点,但siRNA(small interfering RNA)的定量检测仍是评价RNAi抗病毒效果的瓶颈。为了检测抗CSFV特异siRNA分子(siN1和siN2)在细胞中的表达水平,设计并以交叉组合方法筛选了具有较高特异性和灵敏度的siRNA特异茎环引物(SLP-N1-6和SLP-N2-8),成功地建立了最优的siN1和siN2的茎环法RT-qPCR检测方法。该方法表现出良好的特异性和较高的灵敏度,能检测出102至108个拷贝的siRNA,至少可达7个数量级的检测范围,平行性好(Rsq=0.999),扩增效率高(Eff.=98.2%)。茎环法RT-qPCR能准确地定量检测抗CSFV的PK-15细胞克隆的siN1/siN2表达水平,可结合常规的检测病毒水平的间接免疫荧光和TCID50等技术定量评价RNAi抗CSFV的有效性,为未来抗猪瘟转基因猪的抗病毒效果评价提供了先进的检测技术。     

基于PCR技术的miRNA定量检测方法

microRNA(miRNA)是一类广泛存在于真核生物中,不编码蛋白质的短序列RNA,它广泛参与真核生物的生长发育、新陈代谢和应激反应等生命活动。但绝大多数miRNA的生物学功能还不清楚,通过灵敏的定量检测方法,了解miRNA在不同组织部位的时空表达,是探究其功能的重要环节。现着重介绍了2类7种基于PCR技术的miRNA定量检测方法的基本原理和实验流程,并分析了这些定量检测技术间的异同和适用范围。     

miR-451a和miR-21-5p双重实时荧光定量PCR检测体系的建立及应用

目的 为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR (real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法 设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果 优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论 该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。     

人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。     

Real-time quantification of microRNAs by stem-loop RT-PCR

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.     

A Facile and Specific Assay for Quantifying MicroRNA by an Optimized RT-qPCR Approach

Background

The spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.

Results

We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.

Conclusions

The cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.     

Real-time quantification of microRNAs by stem–loop RT–PCR

A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.