Site Directed Mutagenesis (SDM) Rat Point mutation Rat-2

Get tips on using Senescence β-Galactosidase Staining Kit - Cell Signaling to perform Reporter gene assay β-galactosidase substrates - MIA PaCa-2

Get tips on using OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) to perform ROS assay cell type - MiaPaCa-2 pancreatic carcinoma

Get tips on using ROS-ID® Total ROS/Superoxide detection kit to perform ROS assay cell type - MiaPaCa-2 pancreatic carcinoma

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform siRNA / RNAi /miRNA transfection Mouse - Lung adenocarcenoma (A549/LTEP-a-2) Lipofectamine

Get tips on using QCM ECMatrix Cell Invasion Assay, 24-well (8 µm), fluorimetric to perform Cell migration / Invasion cell type - SaOS-2

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.

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - U937 MK2 (MAPK Kinase 2) Viral vectors

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - Jurkat MK2 (MAPK Kinase 2) Viral vectors

Get tips on using Rab 7 siRNA (h) to perform siRNA / miRNA gene silencing Human - BEAS-2B RAB7A