Identification of miRNAs in a Liver of a Human Fetus by a Modified Method

Background

miRNAs are 17–25 nucleotides long RNA molecules that have been found to regulate gene expression in human cells. There are studies showing that different groups of miRNAs are involved in development of different tissues. In hepatocytes there are reported particular types of miRNAs that regulate gene expression.

Methods

We established a human fetal liver cDNA library by a modified cloning protocol. Then plasmid isolation from the colonies was performed. After sequencing and database searching, the miRNAs were recognized. RT-PCR and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA.

Results

One novel miRNA was discovered, together with another 35 previously-known miRNAs in the fetal liver. Some of them existed in variants. The miRNAs identified were validated by RT-PCR and sequencing. Quantitative analysis showed that they have variable expression.

Conclusion

Our results indicate that a special group of miRNAs may play an important role in fetal liver development in a synergistic manner.     

小鼠肝脏MicroRNA的提取与分离

本文对小鼠肝脏microRNA进行分离制备及相关性功能研究.采用poly(A)加尾方法,分离提取小鼠肝脏中的microRNA;利用T4 RNA连接酶,在microRNA两端加上连接引物,通过RT PCR制备microRNA的cDNA文库;经过对cDNA文库的质粒连接,克隆测序获得microRNA. 结果显示：获得4条有效序列,其中包括2条已知序列miR 122和2条未知小分子RNA序列.经查证, 2条microRNA片段在miRBase库中未有记录,在二级结构分析中可形成茎环结构,符合microRNA的特征. BLAST比对显示： 2个序列位于小鼠载脂蛋白B基因和小鼠28S核糖体RNA上,可能与基因调控有关,其功能有待进一步研究.     

T7启动子在哺乳类动物细胞中启动外源基因表达的研究

人低密度脂蛋白（LDL）受体基因cDNA和氯霉素已酞转移酶基因（CAT）及PolyA信号序列被克隆进pGEM4载体的T7噬茵体启动子下游,构建成质粒pT7LDLR和pT7CAT．两个重组质粒转化CHO细胞．PCR和CAT酶实验显示：两个基因被T7噬菌体启动子所启动．结果证实真核生物RNA聚合酶能够识别T7启动子,转录外源基因．常用的含有T7启动子的质粒可同时作为原核生物和真核生物的表达载体．     

大肠杆菌poly(A)化mRNA基因的芯片制备

利用大肠杆菌mRNA中存在的一定程度的poly(A)现象,利用oligo(dT)与poly(A)特异结合的特性,纯化并逆转录mRNA,并应用RD-PCR双方法获得了170多条大肠杆菌poly(A)化mRNA的基因片段,利用这些片段打印成基因芯片,以供后续大肠杆菌的基因表达研究。     

人Ⅱ型囊泡单胺转运体基因的克隆

目的 为获得人Ⅱ型囊泡单胞转运体（vesicular monomine transporter-2VMAT2)以治疗帕金森病,克隆添加Kozak序列的VMAT2cDNA,用于真核细胞表达。方法 从人胎脑中提取总RNA,逆转录成cDNA,设计一对引物用PCR方法扩增目的cDNA片段并将其重级一起pGEM-Easy-T载体中,酶切鉴定插入片段后,进行全序列测定。结果 RT-PCR扩增到1637bp的带有Kozak序列的cDNA片段。结论 克隆人VMAT2cDNA片断,可用于真核细胞表达。     

人脑内-含有ACP样结构域新基因的发现

为寻找脑内新基因,以正常成人全脑cDNA为模板,采用锚定PCR方法进行扩增,将经琼脂糖DNA电泳 鉴定获得的一约1200bp大小的特异性条带回收,并克隆入Teasy载体.用310 Genetic Analyzer进行自动测序. 所得序列进行生物信息学分析BLAST相似性分析结果证明所得序列为新序列,读框分析表明,该序列中存在 一完整编码区,编码含357个氨基酸的蛋白质.ProDom软件分析发现其含有酰基携带蛋白(ACP)样结构域. 随后,经3'RACE法克隆到该基因的全长cDNA,其全长为2024bp,染色体定位在14q11.2,含有16个外显子, 15个内含子,该基因已登录到GenBank.经设计编码区引物,从Teasy载体扩增出编码区后再克隆入pGEX-4T1 表达载体,经异丙基硫代-D-乳糖苷(IPTG)化学诱导表达.其编码区克隆人pGEX-4T1表达载体后,转入 JM109宿主菌,经IPTG诱导已得到表达.点杂交及RNA印迹表明,该基因在正常成人脑内广泛高表达.     

Identification and Characteristics of Cattle MicroRNAs by Homology Searching and Small RNA Cloning

MicroRNAs (miRNA) are a class of noncoding RNA molecules that regulate gene expression by an RNA-interfering pathway through cleavage or inhibition of the translation of target mRNA. The 254 cattle miRNA candidates found by homology searching frequently clustered at certain chromosomes, and some are possibly expressed from more than one genomic locus. They were partially verified by cloning from a small cattle RNA library, where 31 distinct miRNAs were identified: 18 previously registered in the database of miRBase, 11 novel and homologous to known mammalian miRNAs, and 2 potentially novel without homology to any known miRNAs. Partial miRNA expression was detected by RT-PCR in cattle tissues, such as brain, liver, lung, and heart; some were expressed in all tissues and others in a specific tissue. Sequence alignments revealed that many had end variants, most of which differed in the 3′ end; a small number differed in the 5′ end. This indicates that the same miRNA gene can be individually modified in the process of miRNA biogenesis and could have a different role in regulating target gene expression.     

Identification of novel stress-regulated microRNAs from Oryza sativa L.

MicroRNAs (miRNAs) are a type of small non-coding RNA found in eukaryotes. They play a key role in gene expression by down-regulating gene expression and are involved in the environment stress response in plants. Although a large number of miRNAs have been identified from Arabidopsis, few studies have focused on Oryza sativa miRNAs, especially on stress-related miRNAs. Five cDNA libraries of small RNAs from rice seedlings treated with cold, dehydration, salinity, and abscisic acid (ABA), as well as wild-type seedlings, were constructed. Seven rice novel miRNAs were identified by Northern analysis, and their expression patterns under different stress treatments were determined. Results showed that the expression of several novel miRNAs was regulated by one or more stress treatments. Our identification of novel stress-related miRNAs in rice suggests that these miRNAs might be involved in rice stress response pathways.     

Facile means for quantifying microRNA expression by real-time PCR

MicroRNAs (miRNAs) are 20-24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. Several Arabidopsis miRNA sequences were tested using SYBR Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having asfew as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.