Q: What is cloned into the miRNA clones?
A: OriGene抯 miRNA clone inserts is composed of pri-mir (60- 70nts) and its 250-300nts flanking genomic sequence on both sides. In general, the flanking sequence is required for correct pri-mir expression and mature microRNA processing.
Q: How do I know the miRNA is being expressed?
A: In OriGene, small RNA was isolated from transfected cells. Then RT-PCR or qPCR was used to measure the expression of miRNA level.
Q: Are the miRNA clones fully sequenced? Where can I find the sequence information?
A: Yes, they are. All miRNA clones were sequenced to ensure that the pre-mir sequences match reference sequence in miRBase. Please note that the exact flanking sequence of a miRNA clone may differ from the NCBI reference with respect to biological polymorphisms. This should not affect the function of the mature miRNA.
Q: What sequencing primers should I use?
A: VP1.5, 5'GGACTTTCCAAAATGTCG 3?(Tm=51C) and XL39, 5'ATTAGGACAAGGCTG-TGGG 3'(Tm=60C) can be used to sequence from the 5'and 3'-end of the insert, respectively. Both plasmids are provided with miRNA expression clone. Please note that the two primers usually do not work well for amplification of the insert due to the large difference in their Tms.
Q: How can I monitor the transfection efficiency?
A: In OriGene we monitor tGFP signal for transfection. PCMV6-mir vector contains an IRES-tGFP reporter gene, which is driven by SV40 promoter and is down-stream of neomycin gene. Independent tGFP and pre-mir expression from different promoters is designed to minimize the interference between the two expression cassettes and therefore tGFP can truly reflect the transfection efficiency.
Q: Can I create stable cell line with the miRNA plasmids? What is the selection marker?
A: Yes. You can create the stable line by using G418 selection or sorting the GFP-expressing cells.
Q: Is it true that only one miRNA is expressed in each plasmid?
A: No. Since some (about 10%) miRNA genes are clustered in close region in chromosome and they may be naturally express together. OriGene's miRNA clones didn't separate them. For all the miRNA clones that contain more than one miRNA, they are listed on www.origene.com and linked to the same miRNA clones.
Q: How is the miRNA vector validated?
A: All OriGene's miRNAs match reference sequence in miRBase. Always note that the exact flanking sequence of a miRNA clone may differ from the NCBI reference with respect to biological polymorphisms.
Q: What is the control plasmid for miRNA expression plasmid?
A: In OriGene, we use pCMV6-mir vector as control in testing miRNA expression.
Q: Do the miRNA plasmids come with any controls?
A: No. pCMVMIR vector can be purchased (cat# PCMVMIR).
Q: How do I cite this product?
A: We recommend that you refer to the product by its specific catalog number and refer to us as OriGene Technologies (Rockville, MD). Furthermore, we'd love to hear from you when your paper is published. Inform us and we will send a gift.