shRNA gene silencing Human - Neuroblastoma cells (SH-SY5Y) Beclin 1

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 has 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 the efficacy of transduction and shRNA on its target site.

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4 years ago

4 years ago by Eufrosina Sagese Italy

Is a knockdown using shRNA permanent?

Is a knockdown using shRNA permanent and if not is there a known duration?

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Found 1 matching solution for this experiment

pSilencer 2.1-U6 Hygro

Thermo Fisher Scientific

Protocol tips
SH-SY5Y, Dulbecco’s modified Eagle’s medium (DMEM, 12100-046; Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS, SH30088.03; Thermo Scientific HyClone), 100 U/ml penicillin, 100 µg/ ml streptomycin and 2 mM glutamine. Cells were maintained in a humidified 10% CO2 atmosphere at 37°C. A human Beclin 1 shRNA hairpin (target sequence: CTC AGG AGA GGA GCC ATT T) was cloned into the HindIII and BamH1 sites of pSilencer 2.1-U6 Hygro. Beclin 1 shRNA and control scrambled shRNA plasmids were transfected into SH-SY5Y cells and selected with 50 µg/ml Hygromycin.
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