shRNA gene silencing Mouse Prostate cancer cell lines (DU145 and PC3) CD24

Get tips on using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit to perform Cell line authentication Human prostatic cancer cell line PC3

Get tips on using MCM4 shRNA (h) Lentiviral Particles to perform shRNA gene silencing Human - SiHa MCM4

Get tips on using CD24 Monoclonal Antibody (eBioSN3 (SN3 A5-2H10)), PE, eBioscience™ to perform Flow cytometry Anti-bodies Human - CD24

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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

Get tips on using TLR10 shRNA (h) Lentiviral Particles to perform shRNA gene silencing Human - THP-1 TLR10

Get tips on using TLR10 shRNA (h) Lentiviral Particles to perform shRNA gene silencing Human - THP-1 TLR10

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

Get tips on using GATA-1 shRNA Plasmids (h) to perform shRNA gene silencing Human - TF‐1 GATA‐1

Get tips on using connexin 43 ShRNA to perform shRNA gene silencing Human - Neuroblastoma cells (SH-SY5Y) Connexin 43 lentiviral particles