protein-expression-and-purification-mammalian-cells-hek-293-mt-pa

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.

Get tips on using heat shock protein family A (Hsp70) member 5 to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) HSPA5 (GRP78)

Get tips on using Mouse C Reactive Protein ELISA Kit (PTX1) (ab157712) to perform ELISA Mouse - C-Reactive Protein/CRP

Get tips on using Rat Bone Morphogenetic Protein 2 ELISA to perform ELISA Rat - BMP-2

Get tips on using Anti-Prosurfactant Protein C (proSP-C) Antibody to perform Flow cytometry Anti-bodies Mouse - proSP-C

Get tips on using FuGENE® 6 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat hepatic stellate cells

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat hepatic stellate cells

Get tips on using QIAGEN Proteinase K (10 ml) to perform RNA isolation / purification Cells - primary mouse morula cells

Generally isolating RNA from Gram-negative bacteria is easy, however keeping your working environment clean and RNase free (use RNase inhibitor) is essential. Some common points to keep in mind: a) Use fresh samples for isolation or store them by freezing in RNA stabilizing buffer until use. b) Choose the bacterial input amounts carefully, to ensure buffer volumes are adequate and not to overload the columns.