FAQ: miRNA Analysis
Q: If the specific miRNA precursor I am studying is not in your Precursor Clone Collection, can you clone it for me?
A: We do not offer cloning services for miRNA or any other vectors. However, we have convenient cloning and expression vectors that you may use to clone in your own sequence. These vectors will accommodate precursor sequences from any species.
A: Our miRNA expression vectors may be used in either transient transfection or stable clone selection. The vectors are provided as mammalian transient transfection vectors containing either puromycin (pEP backbone) or GFP-puromycin fusion (pEGP backbone). After transfection, you can select the stable cell lines by culturing cells in medium containing 1-2 µg/mL puromycin. These stable cell lines will stably express the miR gene as well as the puromycin or GFP-puromycin marker.
Q: I’m studying human miRNAs. You don’t have the human miRNA sequence I’m looking for, but you do have the mouse sequence. Are they the same?
A: Sequence homology of specific miRNAs appears to be conserved in human and mouse in the mature form only. Most of the time the precursor sequences are slightly different.
Q: I have different pre-miRNA sequences that I would like to test for expression. Can I clone multiple sequences together into your miRNA expression vector?
A: We have not specifically tested our miRNA expression vectors with multiple pre-miRNA sequences, but it may be possible.
Q: Why do your miRNASelect™ Expression Vectors contain an EF-1 promoter instead of the more common CMV promoter?
A: When making stable expression, CMV can become weaker and weaker as methylation occurs in the CMV promoter region. Using the EF-1 promoter overcomes this issue.
Q: What is the structure of the sequences found in your miRNASelect™ Precursor Vectors?
A: The miRNA precursor cloned into our vectors consists of the stem loop structure plus an additional 100 bp on each side of the stem loop. This maintains the miRNA precursor structure for better miRNA processing.