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.     

The use of miRNAs as reference genes for miRNA expression normalization during <Emphasis Type="Italic">Lilium</Emphasis> somatic embryogenesis by real-time reverse transcription PCR analysis

Expression profiling of miRNAs has the ability to reveal the essence of somatic embryogenesis (SE). qRT-PCR is one of the most commonly used techniques for dynamic miRNA detection but requires optimal reference genes for data reliability. This is the first report on reference gene validation for miRNA expression normalization in Lilium (Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor). In this study, seventeen miRNAs together with two snRNAs (U4, U6), one rRNA (5S rRNA) and three protein-coding genes (FP, ACT, GAPDH) were selected as reference candidates, and their expression stability was validated by qRT-PCR among eleven developing SE cultures in two lilies. Four normalization algorithms, including geNorm, BestKeeper, NormFinder and RefFinder, were also used to evaluate the stability of the reference candidates. For Lilium pumilum DC. Fisch., lpu-miR159a was the optimal reference gene during SE, followed by lpu-miR408b, while U6 was the least stable reference candidate. For Lilium davidii var. unicolor, FP presented greater stability than did half of the miRNA candidates, but the best reference gene was lda-miR162, followed by lda-miR159a. Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies. In general, miRNAs are superior to common protein-coding genes and snRNAs / rRNAs as reference genes for miRNA expression normalization during Lilium SE, and the most suitable reference miRNA is different between two species in the same Lilium genus. This is a pioneer study using suitable miRNAs as reference genes in Lilium and constitutes a small but essential step for the further exploration of miRNA function in Lilium, thus offering valuable references for other plants.     

Identification of mouse serum miRNA endogenous references by global gene expression profiles

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.     

Global analysis of non-coding small RNAs in Arabidopsis in response to jasmonate treatment by deep sequencing technology

In plants, non-coding small RNAs play a vital role in plant development and stress responses. To explore the possible role of non-coding small RNAs in the regulation of the jasmonate (JA) pathway, we compared the non-coding small RNAs between the JA-deficient aos mutant and the JA-treated wild type Arabidopsis via high-throughput sequencing. Thirty new miRNAs and 27 new miRNA candidates were identified through bioinformatics approach. Forty-nine known miRNAs (belonging to 24 families), 15 new miRNAs and new miRNA candidates (belonging to 11 families) and 3 tasiRNA families were induced by JA, whereas 1 new miRNA, 1 tasiRNA family and 22 known miRNAs (belonging to 9 families) were repressed by JA.     

MicroRNA与药用植物

MicroRNA（miRNA）是一类长度约为19～25个核苷酸的内源性非编码小分子单链RNA,对植物生长发育、次生代谢产物的生物合成以及抗逆性等有重要作用.近年来关于植物中miRNA的研究进展异常迅速,尤其是药用植物,目前已从一些药用植物中鉴定出miRNA,并分析了其在药用植物中的调控作用,这对药用植物的研究具有重要意义.本文综述了miRNA在植物中的产生途径、作用机制,同时重点介绍了miRNA在药用植物中的研究情况,使人们对药用植物中miRNA有更深的认识,为药用植物研究拓展新思路.     

The discovery approaches and detection methods of microRNAs

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition. Computer-based approaches for miRNA gene identification are being considered as indispensable in miRNAs research. Similarly, experimental approaches for detection of miRNAs are crucial to the testing and validating of computational algorithms. The detection of miRNAs in tissues or cells can supply valuable information for investigating the biological function of these molecules. Selective and highly sensitive detection methods will pave the way for extended understanding of miRNA function within organisms. In this review, we summarize the various computational methods for identification of miRNAs as well as the methodologies that have been developed to detection miRNAs.     

Comparison of normalization methods with microRNA microarray

MicroRNAs (miRNAs) are a group of RNAs that play important roles in regulating gene expression and protein translation. In a previous study, we established an oligonucleotide microarray platform to detect miRNA expression. Because it contained only hundreds of probes, data normalization was difficult. In this study, the microarray data for eight miRNAs extracted from inflamed rat dorsal root ganglion (DRG) tissue were normalized using 15 methods and compared with the results of real-time polymerase chain reaction. It was found that the miRNA microarray data normalized by the print-tip loess method were the most consistent with results from real-time polymerase chain reaction. Moreover, the same pattern was also observed in 14 different types of rat tissue. This study compares a variety of normalization methods and will be helpful in the preprocessing of miRNA microarray data.