MiRNa- SiRNA - Micro RNA

Integrated Probe Design and Novel Labeling Method Provides for Optimal Sensitivity and Specificity Using Total RNA Samples.

SANTA CLARA, Calif. -- Agilent Technologies Inc. (NYSE:A) today introduced a microarray-based assay for microRNA (miRNA) expression profiling, delivering new levels of sensitivity and specificity while incorporating simple protocols for labeling and detection. The assay allows for very small amounts of sample (100 ng of total RNA), making it well-suited for miRNA expression profiling experiments.

miRNAs, a class of small non-coding RNAs that are only 19-30 nucleotides long, are estimated to regulate approximately 30 percent of all human genes. They were little understood five years ago, but now there are approximately 500 known miRNAs in humans, with ongoing discovery directed toward an estimated 10,000.

Recent studies have shown that distinct miRNA expression patterns are associated with a number of tumor types as well as involved in regulating processes such as cell development, metabolism and viral infections. Because miRNAs are potential regulators of gene expression, scientists are increasingly interested in measuring them for research, drug discovery, other therapeutics and, eventually, diagnostic tests.

"miRNA profiling is a perfect application for the Agilent microarray platform," said Yvonne Linney, Agilent general manager, Genomics. "Our flexible manufacturing procedure allows us to add new content as new miRNAs are discovered; the multipack format makes this an economical platform; and the high sensitivity achieved with the system enables researchers to detect miRNAs from very small amounts of starting material. This application is an important addition to our microarray portfolio, and is synergistic with Agilent offerings for gene expression studies and array-based CGH."

The Agilent miRNA system has been in use by several early access labs around the world over the past six months. Agilent Laboratories developed the chemistry and probe design, which are used with Agilent's SurePrint in situ synthesis microarray fabrication platform to provide the flexibility and sensitivity demanded by this fast-moving area of research. This is a good example of how Agilent Labs collaborates with the company's commercial businesses to generate innovative solutions.

"Agilent's platform for miRNA expression profiling includes a straightforward and easy sample-preparation procedure combined with their well established ink-jet printed oligonucleotide arrays," said Zora Modrusan, scientist and head of the Microarray Laboratory, Molecular Biology at Genentech. "An advantage of their technology compared to the other ones is that a very low amount of starting total RNA sample is required, thus enabling miRNA profiling from clinical samples."

The Agilent miRNA assays are available in multipack format with eight microarrays printed on a standard 1 in. x 3 in. slide, which provides economical cost-per-experiment. The miRNA microarrays are processed using the standard Agilent microarray platform, including hardware and software required for hybridization, scanning, feature extraction and data analysis. One-color analysis further simplifies the experimental design.

"Lung cancer is the leading cause of cancer-related deaths in Japan," said Dr. Takashi Takahashi, professor of Oncology, Molecular Carcinogenesis at Nagoya University. "We have shown for the first time that let-7 expression is frequently reduced in lung cancers and that alterations in the miRNA expression may have a prognostic impact on the survival of surgically treated lung cancer patients. Agilent miRNA arrays give us the comprehensive miRNA expression profile with excellent performance on sensitivity and accuracy. I expect that the studies of Agilent miRNA array may ultimately provide a foundation for a new paradigm of the involvement of miRNA in human oncogenesis."

Agilent is a leading worldwide provider of microarray-based genomics solutions. It provides research tools that enable scientists to study a wide range of applications including gene expression, alternative splicing, chromosomal aberrations and gene copy number (aCGH), protein/DNA interactions (ChIP on Chip) and DNA methylation. For further details about Agilent's microarray solutions, visit www.OpenGenomics.com.

About Agilent Technologies

Agilent Technologies Inc. (NYSE:A) is the world's premier measurement company and a technology leader in communications, electronics, life sciences and chemical analysis. The company's 19,000 employees serve customers in more than 110 countries. Agilent had net revenue of $5.0 billion in fiscal year 2006. Information about Agilent is available on the Web at www.agilent.com.

Labels: agilent, CGH, DNA, genomics, microarray, miRNA, miRNA labelling, RNA, siRNA

If you like this post Subscribe to my Blog Feed

Share It : Digg  | Sphinn  | Netscape  | Google  | reddit  | del.icio.us  | Squidoo  | StumbleUpon  | Yahoo MyWeb  

In genetics, microRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length regulating gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down regulate gene expression. They were first described in 1993 by Lee and colleagues, yet the term microRNA was only introduced in 2001 in a set of three articles in Science magazine. (26 October 2001).

Formatting and Processing of miRNA ( MicroRNA )

The genes encoding miRNA s are much longer than the processed mature miRNA molecule; miRNA s are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Micro processor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to their lack of Drosha homologs; instead, Dicer homologs alone effect several processing steps.

Zeng et al. have shown that efficient processing of pre-miRNA by Drosha requires presence of extended single-stranded RNA on both 3'- and 5'-ends of hairpin molecule. They demonstrated that these motifs could be of different composition while their length is of high importance if processing is to take place at all. Their findings were confirmed in another work by Han et al. Using bioinformatical tools Han et al. analysed folding of 321 human and 68 fly pri-miRNA s. 280 human and 55 fly pri-miRNA s were selected for further study, excluding those molecules which folding showed presence of multiple loops. All human and fly pri-miRNA contained very similar structural regions, which authors called 'basal segments', 'lower stem', 'upper stem' and 'terminal loop'. Based on the encoding position of miRNA, i.e. in the 5'-strand (5'-donors) or 3'-strand (3'-donors), thermodynamical profiles of pri-miRNA were determined. Following experiments have shown that Drosha complex cleaves RNA molecule ~2 helical turns away from the terminal loop and ~1 turn away from basal segments. In most analysed molecules this region contains unpaired nucleotides and the free energy of the duplex is relatively high compared to lower and upper stem regions.

Most pre-miRNA s don't have a perfect double-stranded RNA (dsRNA) structure topped by a terminal loop. There are few possible explanations for such selectivity. One could be that dsRNAs longer than 21 base pairs activate interferon response and anti-viral machinery in the cell. Another plausible explanation could be that thermodynamical profile of pre-miRNA determines which strand will be incorporated into Dicer complex. Indeed, aforementioned study by Han et al. demonstrated very clear similarities between pri-miRNAs encoded in respective (5'- or 3'-) strands.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5' end. The remaining strand, known as the anti-guide or passenger strand, is degraded as a RISC complex substrate. After integration into the active RISC complex, miRNA s base pair with their complementary mRNA molecules and induce mRNA degradation by argonaute proteins, the catalytically active members of the RISC complex. It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process is not coupled to ongoing protein translation from the mRNA.

Labels: genetics, microRNA, miRNA, miRNA labelling, mRNA, RNA, siRNA

If you like this post Subscribe to my Blog Feed

Share It : Digg  | Sphinn  | Netscape  | Google  | reddit  | del.icio.us  | Squidoo  | StumbleUpon  | Yahoo MyWeb