crispr-mouse-deletion-raw-264-7-dcstamp

Get tips on using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit to perform Cell line authentication MCF-7 cell line

Get tips on using Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham to perform Mammalian cell culture media MCF-7

Get tips on using MammoCult™ Human Medium Kit to perform 3D Cell Culture Media Human breast cancer MCF-7 cells-Mammospheres

Get tips on using CpGenome Universal DNA Modification Kit to perform DNA methylation profiling Gene specific profiling - MCF-7 Estrogen receptor alpha

Get tips on using ON-TARGETplus Human NBN (4683) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MCF-7 NBS1/NBN

Get tips on using GenJet™ In Vitro DNA Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines MCF-7

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.

Get tips on using DMEM/F-12, no phenol red to perform 3D Cell Culture Media Human breast cancer MCF-7 cells-Mammospheres

Get tips on using Live/Dead Cell Double Staining Kit to perform Live / Dead assay mammalian cells - MCF-7 human breast cancer cells

Get tips on using Oris™ Universal Cell Migration Assembly Kit, 96 wells to perform Wound healing assay cell type - human MCF-7