Controls for Overexpressing miRNAs - (Jun/09/2009 )
I am going to overexpress miRNAs in mammalian cells by cloning the miRNAs into the vector pcDNA 6.2GW/EMGFP-miR (Block-it Pol LL miR RNAi Expression Vector kit) (Invitrogen). The kit offers a negative control that has an insert capable to be processed into mature miRNA, but predicted not to target an known vertebrate gene.
It also offers a positive control in which has been cloned with a miRNA targeting to lacZ gene. Co-transfection of this positive control with another vector (pcDNA1.2/V5-GW) expressing a lacZ gene followed by western blot or staining methods to assess knockdown of beta-galactosidase.
However, we have borrowed the vector pcDNA6.2GW/EMGFP and we don't have pcDNA1.2/V5-GW. I am thinking if I can clone the lacZ gene into another vector, e.g. pcDNA3.1-His tagged and then do western blot to assess the knockdown by anti-His antibody or not. Any comments?
Actually, do you think there is a need to do this positive control (Co-transfection of vectors containing the miR-lacZ and lacZ gene)? In some papers, people use transfecting miR-lacZ expressing vector to show they do not have non-specific RNAi. Quite confused.
Thank you very much.
The pcDNA 6.2GW/EMGFP-miR is an shRNA expression vector using polII promoter with mir-155 flanking sequences. Therefore, all the positive and negative controls are "shRNAs", not "miRNAs".
So, you want to express "miRNA" or "shRNA via a miRNA template"? They are not the exactly same setup for cloning and analysis. Also, if you want to express shRNA, just use a polIII promoter; if you want to express miRNA, just clone miRNA with its flanking sequences into, for example pcDNA3.1 or lenti vectors.
By the way, Biosettia sells lentivirus ready miRNA, SBI sells lenti miRNA vectors, and others like Ambion sells chemical synthesis miRNA.
I want to express a human miRNA and I already cloned a genomic region with the pre-miRNA into a pcDNA3.1/V5-His-Topo. I checked the miRNA overexpression by RT-PCR and it was huge and I wanted to transfect my cells but I am unsure about my negative control - I wanted to use the pcDNA3.1/V5-His-Topo with LacZ but it is not really a pre-miRNA sequence, with hairpin structure. Would you suggest cloning a precursor for a miRNA from a different species? Thank you for any tips.