siRNA / miRNA gene silencing Human Jurkat MK2 (MAPK Kinase 2)

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Hello Iam a phd student in pharmacy and i want to know if this technology is suitable to knockout or silencing part of the gas5 gene in BV2 cells please

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Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.

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