3D Cell Culture Media

Get tips on using SNP Type™ 96.96 Genotyping Reagent Kit with Control Line Fluid—10 IFCs to perform Cell line authentication Human prostatic cancer cell line PC3

Get tips on using celecoxib to perform Autophagy assay cell type - U87MG

Get tips on using Corning® BioCoat™ Matrigel® Invasion Chamber with 8.0 µm PET Membrane in four 6-well Plates to perform Cell migration / Invasion cell type - 4T1

Get tips on using Corning® BioCoat™ Matrigel® Invasion Chambers with 8.0 µm PET Membrane in two 24-well Plates to perform Cell migration / Invasion cell type - BxPC-3

Get tips on using Corning® BioCoat™ Matrigel® Invasion Chambers with 8.0 µm PET Membrane in two 24-well Plates to perform Cell migration / Invasion cell type - MG-63

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.

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.

Get tips on using Torin 1 to perform Autophagy assay cell type - Mel cells

Get tips on using Atg3 Antibody to perform Autophagy assay cell type - KG1 cells

Get tips on using Dansylcadaverine to perform Autophagy assay cell type - C6