小桐子低温胁迫下microRNA实时荧光定量PCR内参的筛选与比较

选择合适的内参对实时荧光定量PCR(qRT-PCR)结果的准确性极其重要,在microRNA(miRNA)的qRT-PCR分析中尤为如此。通过筛选适宜分析小桐子低温胁迫下miRNA定量表达的内参,为小桐子及其他物种mi RNA的qRT-PCR分析提供有用的理论参考。基于以前的小RNA-seq结果,以低温处理的小桐子为材料,挑选11个候选内参基因,用实时荧光定量PCR技术检测它们在不同样本中的表达量,采用GeNorm、NormFinder和BestKeeper软件进行表达稳定性综合分析。结果表明,表达最稳定的基因是miR6448和U6,miR6448的Ct值为23左右,表达丰度适中;U6的Ct值为10左右,表达丰度高;因此,miR6448可作为小桐子低温胁迫下表达丰度适中的miRNA qRT-PCR的内参基因;U6可作为小桐子低温胁迫下丰度较高的miRNA qRT-PCR的内参基因。     

版纳微型猪近交系TNF-α基因mRNA实时荧光定量PCR检测方法的建立

目的快速、灵敏、可靠的检测与临床多种疾病密切相关的重要基因TNF-α的表达情况,构建TNF-α基因及内参GAPDH基因荧光定量PCR质粒标准品。方法利用版纳微型猪近交系4～6月龄猪建立动物模型,提取皮肤创面总RNA,设计特异引物,进行RT-PCR扩增。纯化目的片段与pMD18-T载体连接,转化宿主菌DH5α,提取重组质粒DNA,并经酶切、PCR和测序鉴定,计算重组质粒原液拷贝数浓度并制备梯度浓度标准品,进行实时荧光定量PCR,生成标准曲线。结果建立的TNF-α基因和GAPDH内参基因mRNA表达实时荧光定量PCR检测方法灵敏度分别可达103和105拷贝,线性范围分别为103～109和105～109拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间存在的线性关系R2分别为0.993和0.999,扩增效率E分别为111.073%和95.948%。结论成功的构建了版纳微型猪近交系TNF-α基因质粒标准品和标准曲线,并用内参基因GAPDH进行校正,此方法可为探讨TNF-α基因在临床多种疾病中所发挥的分子机理奠定基础。     

实时荧光定量PCR中内参基因的选择

实时荧光定量PCR技术是分析基因表达谱的一种常用方法,在分析中选择合适的内参基因对数据进行校正是得到可信数据的关键。以Lactobacillus helveticus H9为研究对象,应用实时荧光定量PCR技术,评价了5种常用内参基因ldh、recA、rpoB、gapdh和16S rRNA的表达稳定性,通过geNorm和NormFinder程序进行数据分析,结果表明5个候选内参基因在菌株不同的发酵时间点表达相对都较为稳定,结合两种分析得到其中最为稳定的基因是ldh,适合于用作后续实时荧光定量PCR试验中的内参基因。     

华细辛实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是准确分析目标基因表达水平变化的重要条件。选取SAND-1、ACT、18Sr RNA、CYP2、GAPDH、TUB 6个常用的内参基因作为候选内参基因,利用实时荧光定量PCR技术,通过ge Norm、Norm Finder、Best Keeper、Delta CT等软件分析和评价6个候选内参基因在华细辛营养生长期、花期和花后期等不同生长阶段的根、根茎、叶片、叶柄等不同组织中的表达稳定性,筛选适宜华细辛不同生长阶段不同组织基因表达水平分析的内参基因。结果表明,18S r RNA在华细辛所有样品中表达最为稳定,是进行华细辛基因表达水平分析的适宜的内参基因。研究结果可为华细辛重要活性成分生物合成途径相关基因的功能分析,以及基因差异表达的研究提供有效的校正工具,确保基因表达分析结果的准确性与可靠性。     

内参基因加标法定量土壤微生物目标基因绝对拷贝数

【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。     

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。     

茶树实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是提高实时荧光定量PCR分析(qRT-PCR)准确性的先决条件。该文以茶树(Camellia sinensis) 芽、叶、幼根、嫩茎、花瓣、种子和愈伤组织为材料, 应用实时荧光定量PCR技术, 分析了18S rRNA、GAPDH、β-actin和α-tubulin 4个常用内参基因在茶树不同器官组织中的表达情况。经GeNorm和NormFinder软件分析发现, 当利用荧光定量PCR分析比较茶树不同器官组织中的基因表达差异时, 可选择β-actin作为校正内参基因; 而比较不同成熟度的叶片和愈伤组织时, 可以选择GAPDH作为校正内参基因。     

茶树实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是提高实时荧光定量PCR分析（qRT-PCR）准确性的先决条件。该文以茶树（Camellia sinensis）芽、叶、幼根、嫩茎、花瓣、种子和愈伤组织为材料,应用实时荧光定量PCR技术,分析了18S rRNA、GAPDH、β-actin和α-tubulin4个常用内参基因在茶树不同器官组织中的表达情况。经GeNorm和NormFinder软件分析发现,当利用荧光定量PCR分析比较茶树不同器官组织中的基因表达差异时,可选择β-actin作为校正内参基因;而比较不同成熟度的叶片和愈伤组织时,可以选择GAPDH作为校正内参基因。     

小菜蛾U6 snRNA基因的克隆及作为miRNA定量表达分析内参基因的评价

【目的】克隆小菜蛾Plutella xylostella (L.)小分子非编码RNA U6的cDNA序列,并评价其是否适合作为定量检测小菜蛾microRNA (miRNA)表达量的内参基因。【方法】本研究采用RT-PCR 克隆获得了小菜蛾4龄幼虫核小RNA(small nuclear RNA, snRNA) U6的cDNA序列,并用定量PCR法检测了U6及8种miRNAs在小菜蛾不同发育阶段及不同杀虫药剂处理后的表达稳定性。【结果】小菜蛾U6的cDNA序列全长 94 bp,与其他昆虫U6的核苷酸序列一致性达98.9%。用geNorm和RefFinder软件分析荧光定量PCR结果表明,U6在小菜蛾卵、1-4龄幼虫、蛹和成虫7个不同发育阶段表达稳定;用马拉硫磷、毒死蜱、辛硫磷、灭多威、呋喃虫酰肼、高效氯氰菊酯、氯虫苯甲酰胺、溴虫腈和Bt 9种不同作用机理的杀虫药剂处理3龄末幼虫48 h,对U6的表达水平无显著影响。【结论】小菜蛾U6表达水平不受不同发育阶段和不同杀虫药剂处理的影响,符合作为内参基因的基本特点,可作为定量PCR法评价小菜蛾miRNA或其他非编码小分子RNA表达水平的内参基因。研究结果为小菜蛾miRNA表达水平的准确定量奠定了基础。     

杂交兰实时荧光定量PCR内参基因的筛选

为了给杂交兰的基因功能表达和调控研究提供内参基因,本研究采取同源克隆方法和RT-PCR技术,以杂交兰‘黄金小神童’叶片为材料,分离杂交兰Ch18S r RNA、Ch28S r RNA、Ch ACT、Ch TUA、Ch TUB、Ch UBQ、Ch EF-1α和Chrpo B基因的片段,以10个不同品种杂交兰叶片及‘黄金小神童’花器官不同部位为材料,利用实时荧光定量PCR(Real-time quantitative PCR,q PCR)检测分析各引物的扩增效率和相对表达量,采用ge Norm、Norm Finder和Best Keeper软件对各个候选内参基因的表达稳定性进行分析,并通过研究杂交兰花器官不同部位Ch PDS基因的表达模式来验证筛选得到的内参基因的可靠性。结果显示,各引物扩增的片段长度分别为171 bp、148 bp、183 bp、146 bp、130 bp、147 bp、203 bp、139 bp,扩增效率分别为1.94、2.19、1.88、1.99、2.12、2.20、2.11、1.98。综合3个软件的评价结果发现,杂交兰不同品种叶片中最佳内参基因为Ch ACT,不同花器官部位中最稳定内参基因为Ch ACT、Ch TUB、Ch UBQ和Ch EF-1α;而对于实验中所有样品来说,内参基因稳定性最高的为Ch TUB;说明不同的实验条件下,所需的内参基因不同。Ch PDS基因相对表达水平分析结果证实了所筛选内参基因的可靠性,以Ch UBQ、Ch EF-1α、Ch TUB、Ch ACT及Ch UBQ和Ch EF-1α基因组合进行校正的Ch PDS基因的相对表达量均为花瓣唇瓣蕊柱。     

Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues

Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.     

麦长管蚜miRNA实时荧光定量PCR内参基因的筛选

【目的】筛选特定条件下麦长管蚜Sitobion avenae稳定表达的微小RNA(miRNA)表达分析内参基因。【方法】根据麦长管蚜miRNA Illumina测序结果筛选出miR-10-3p, miR-993, miR-276, miR-275, miR-252a, miR-1, miR-375, pc-15, pc-73和1个常用内参基因U6共10个候选内参基因。利用qRT-PCR测定10个候选内参基因在麦长管蚜有翅蚜和无翅蚜不同发育时期、不同组织以及4种化学药剂(95.1%吡虫啉原药、97.8%噻虫啉原药、95%阿维菌素原药和40%氧乐果乳油)处理无翅蚜中的相对表达量。利用GeNorm, NormFinder, ΔCt法, BestKeeper和RefFinder对10个候选内参基因的表达稳定性进行评价。【结果】qRT-PCR结果表明,在麦长管蚜有翅蚜不同发育阶段、无翅蚜不同发育阶段、有翅蚜和无翅蚜不同组织中和4种药剂处理的无翅蚜中表达量较高的内参基因分别是miR-276, miR-276, miR-10-3p和miR-10-3p;5种方法综合分析得出,在上述4种条件下表达稳定性都较好的内参基因分别是miR-252a, miR-993, miR-275和miR-993。GeNorm软件分析表明不同条件下麦长管蚜内参基因的较佳数目为2,结合RefFinder稳定性综合排序的结果分析可知在有翅蚜不同发育阶段、无翅蚜不同发育阶段、有翅蚜和无翅蚜不同组织中和不同药剂处理无翅蚜中稳定表达的内参基因组合分别是miR-252a和miR-276, miR-993和miR-276, miR-275和miR-10-3p, miR-993和miR-10-3p。【结论】麦长管蚜有翅蚜不同发育阶段、无翅蚜不同发育阶段、有翅蚜和无翅蚜不同组织中和不同药剂处理无翅蚜中内参基因优化组合分别为miR-252a和miR-276, miR-993和miR-276, miR-275和miR-10-3p, miR-993和miR-10-3p。研究结果为麦长管蚜miRNA基因定量表达的研究提供了内参基因选择的依据。     

Identification and Validation of Reference Genes for qPCR Detection of Serum microRNAs in Colorectal Adenocarcinoma Patients

Serum microRNAs (miRNAs) have become a highlighted research hotspot, especially for their great potential as a novel promising non-invasive biomarker in cancer diagnosis. The most frequently used approach for serum miRNAs detection is quantitative real time polymerase chain reaction (qPCR). In order to obtain reliable qPCR data of miRNAs expression, the use of reference genes as endogenous control is undoubtly necessary. However, no systematic evaluation and validation of reference genes for normalizing qPCR analysis of serum miRNAs has been reported in colorectal adenocarcinoma. We firstly profiled pooled serum of colorectal adenocarcinoma, colorectal adenoma and healthy controls and selected a list of 13 miRNAs as candidate reference genes. U6 snRNA (U6) and above-mentioned 13 miRNAs were included in further confirmation by qPCR. As a result, 5 miRNAs (miR-151a-3p, miR-4446-3p, miR-221-3p, miR-93-5p and miR-3184-3p) were not detected in all samples and 2 miRNAs (miR-197-3p and miR-26a-5p) were relatively low with median Cq more than 35, and were excluded from further stability analysis. Then variable stability of other 6 miRNAs (miR-103b, miR-484, miR-16-5p, miR-3615, miR-18a-3p and miR-191-5p) and U6 were evaluated using two algorithms: geNorm and NormFinder which both identified miR-191-5p as the most stably expressed reference gene and selected miR-191-5p and U6 as the most stable pair of reference genes. After validating in an independent large cohorts and selecting miR-92a-3p as target miRNA to evaluate the effect of reference gene, we propose that combination of miR-191-5p and U6 could be used as reference genes for serum microRNAs qPCR data in colorectal adenocarcinoma, colorectal adenoma and healthy controls.     

Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r² = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.     

The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing

Intron removal during pre-messenger RNA (pre-mRNA) splicing involves arrangement of snRNAs into conformations that promote the two catalytic steps. The Prp19 complex [nineteen complex (NTC)] can specify U5 and U6 snRNA interactions with pre-mRNA during spliceosome activation. A candidate for linking the NTC to the snRNAs is the NTC protein Cwc2, which contains motifs known to bind RNA, a zinc finger and RNA recognition motif (RRM). In yeast cells mutation of either the zinc finger or RRM destabilize Cwc2 and are lethal. Yeast cells depleted of Cwc2 accumulate pre-mRNA and display reduced levels of U1, U4, U5 and U6 snRNAs. Cwc2 depletion also reduces U4/U6 snRNA complex levels, as found with depletion of other NTC proteins, but without increase in free U4. Purified Cwc2 displays general RNA binding properties and can bind both snRNAs and pre-mRNA in vitro. A Cwc2 RRM fragment alone can bind RNA but with reduced efficiency. Under splicing conditions Cwc2 can associate with U2, U5 and U6 snRNAs, but can only be crosslinked directly to the U6 snRNA. Cwc2 associates with U6 both before and after the first step of splicing. We propose that Cwc2 links the NTC to the spliceosome during pre-mRNA splicing through the U6 snRNA.     

MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer

Background

MicroRNA (miRNA) is an emerging subclass of small non-coding RNAs that regulates gene expression and has a pivotal role for many physiological processes including cancer development. Recent reports revealed the role of miRNAs as ideal biomarkers and therapeutic targets due to their tissue- or disease-specific nature. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, and laryngeal cancer has the highest incidence in it. However, the molecular mechanisms involved in laryngeal cancer development remain to be known and highly sensitive biomarkers and novel promising therapy is necessary.

Methodology/Principal Findings

To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. The resultant differentially expressed miRNAs were further tested by using quantitative real time PCR (qRT-PCR) on 43 laryngeal tissue samples including cancers, noncancerous counterparts, benign diseases and precancerous dysplasias. Significant expressional differences between matched pairs were reproduced in miR-133b, miR-455-5p, and miR-196a, among which miR-196a being the most promising cancer biomarker as validated by qRT-PCR analyses on additional 84 tissue samples. Deep sequencing analysis revealed both quantitative and qualitative deviation of miR-196a isomiR expression in laryngeal cancer. In situ hybridization confirmed laryngeal cancer-specific expression of miR-196a in both cancer and cancer stroma cells. Finally, inhibition of miR-196a counteracted cancer cell proliferation in both laryngeal cancer-derived cells and mouse xenograft model.

Conclusions/Significance

Our study provided the possibilities that miR-196a might be very useful in diagnosing and treating laryngeal cancer.     

Extended base pair complementarity between U1 snRNA and the 5' splice site does not inhibit splicing in higher eukaryotes, but rather increases 5' splice site recognition

Spliceosome formation is initiated by the recognition of the 5′ splice site through formation of an RNA duplex between the 5′ splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5′ splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5′ splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5′ splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5′ splice site interaction does not decrease splicing efficiency, but rather increases 5′ splice site recognition and exon inclusion. However, low complementarity of the 5′ splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome.