DNA transfection Mammalian cells Primary cells

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Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

Cellular assays Reporter gene assay β-galactosidase substrates U87 and U251 glioblastoma cells

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

Cellular assays Reporter gene assay β-galactosidase substrates human MSCs (mesenchymal stem cells)

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

Cellular assays Reporter gene assay β-galactosidase substrates rat mesenchymal stem cells (MSCs)

Proteins Protein Expression Prokaryotic cells E. coli Integrin αV

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.

RNA shRNA gene silencing Human Neuroblastoma cells (SH-SY5Y) Connexin 43 lentiviral particles

Get tips on using GenElute™ Mammalian Total RNA Miniprep Kit to perform RNA isolation / purification Tissue - Rat Ovaries

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Get tips on using FlashTag™ Biotin HSR RNA Labeling Kits to perform Microarray RNA amplification & Labeling - Rat primary vascular smooth muscle cells Biotin

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Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay mammalian cells - rat cardiomyocytes

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Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay mammalian cells - rat MSC

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Get tips on using Live/Dead cell Staining Kit II to perform Live / Dead assay mammalian cells - mouse iPSC

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