About miRNAs

Increasing evidence indicates that miRNAs regulate complex and diverse pathways governing cell proliferation and cell death, early development, apoptosis, cell differentiation and fat metabolism and suggests links to cancer, neurodevelopment and viral disease. A single miRNA can orchestrate multiple pathways involving the regulation of multiple genes. Conversely, several different miRNAs can cooperatively control a single miRNA target. It is this broad functionality that leads to the complexity of translational control of some genes and the remarkably complex network mediating many facets of eukaryotic cell function. At least 2 independent studies have predicted that 20-30% of human genes could be controlled by miRNAs. They may exist about 300 unique miRNAs in the human and mice genomes with many more to be discovered in the future.

miRNAs are encoded in loci other than their targets and a majority (about 70%) of mammalian miRNA genes are located in defined transcription units. They are transcribed by RNA polymerase II and their activation involves a multi-step process. They regulate gene expression by binding to imperfectly matched binding sites in the 3'-untranslated regions of target mRNAs. They are expressed as longer hairpin molecules, and like siRNAs are generated by DICER, are identical in size (~22 nt), and can associate with RISC in cells. However, miRNAs are different in that non-complementarity exists between the center of the miRNA and the targeted miRNA. Hence, it is the degree of complementarity to its target binding site that determines whether it will function as an miRNA or an siRNA. These tiny ~22nt RNAs therefore induce miRNA degradation or translational repression or both. Understanding of the miRNA-guided network will open new windows for diagnostics and therapy of many human diseases.

Applications: Cloning miRNA targets and evaluating miRNA regulation Screening putative miRNA target sequences miRNA expression profiling to detect differential miRNA expression across tissues and disease states

GenScript miRNA Services

Sequence-directed cloning:

miRNAs, due to their low abundance, are difficult to isolate and once isolated, these 18-24nt RNAs have to purified, ligated to 5' and 3' adapter sequences and after RT-PCR cloned into vectors and sequenced. In place of this, computational analysis from genomic sequences is often used as a valuable tool that complements cloning. New miRNAs are identified in this manner that should correlate with computational analysis. At GenScript, we offer you an alternate way to clone your sequences of interest with our vector-based miRNA technology. In addition to our existing siRNA construction and the entire spectrum of siRNA vectors that we offer, we have launched a miRNA construction service in the vector of your choice. We can assist you with the construction of the gene sequence that you have chosen and provided us, that can be then cloned into any of our miRNA cloning vectors. The resulting miRNA expressing plasmid is purified and ready to transfect and can be used for a variety of applications such as quantitation using Northern blotting, dot blotting, RNAse protection assay, primer extension analysis, Invader assay and quantitative PCR.

GenScript Vectors:

Our GenScript CMV promoter-based plasmid vector line that we have developed is used for cloning. These vectors are designed for long-term gene silencing experiments in a broad range of mammalian cell lines. It is an offshoot of our CMV-based siRNA vectors and carries an RNA polymerase II-type CMV promoter (human cytomegalovirus immediate-early promoter) and an optimized SV40 polyadenylation signal to drive high-level expression of the hairpin RNA. This vector also features an SV40 promoter that expresses one of three antibiotic resistance genes (hygromycin, neomycin, or puromycin) for long-term antibiotic selection used for stable transfections.

Alternatively, we can synthesize and clone your miRNA sequence into any vector that you choose to send to us. The plasmid is sequenced in the region and sent to you along with the QC documents as is the case with all our services.

Price:

Note: Maxiprep plasmid should be used for transfection. You may order Maxiprep (100 ug) from GenScript for $125 extra.

Specifications and Delivery:

GenScript will synthesize the construct exactly according to your sequence using GenScript technology. The synthesized miRNA construct will be cloned into an miRNA expression vector that you desired and will be confirmed by sequencing. The typical delivery includes around 4 μg of lyophilized plasmid DNA which contains your miRNA construct and sequence data documenting the DNA sequence of each clone. The typical delivery time is about three weeks.

Online Ordering:

Orders can be placed through our regular or toll-free phone numbers, email, fax, or online with a formal PO (Purchase Order) or credit card. We recommend our secure web server. Note: If you only want to order miRNA vectors, please browse our miRNA vectors list. We accept POs and major credit cards . A 7% New Jersey sales tax will be applied to orders shipped to New Jersey. Your credit card will be billed under "GenScript Corp." Click here to download our credit reference form. Our customer service representatives are available 24 hours Monday through Friday. For international orders, we must apply the full charge at the time the order is placed. In the unlikely event that any given order cannot be filled, our guarantee will take the form of a full refund. Order online: https://www.genscript.com/ssl-bin/order_miRNA Order by email: order@genscript.com Order by phone: 1-877-436-7274 (Toll-Free) 1-732-885-9188 Order by fax: 1-732-210-0262 1-732-885-5878