rna-isolation-purification-cells-primary-rat-brain-microvascular-endothelial-cells

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

Get tips on using NucleoBond® RNA/DNA to perform DNA isolation / purification Cells - Primary cells Rat cortical neurons

The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.

Isolating RNA from tissues and paraffin-embedded tissue samples can be challenging due to cross-linking of biomolecules and fragmented nucleic acids. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in presence of RNAse inhibitors. The homogenization process should be carried out on dry ice to maintain the integrity of RNA

Get tips on using PureLink™ RNA Mini Kit to perform RNA isolation / purification Cells - primary rat astrocytes

Get tips on using MagMAX™-96 Total RNA Isolation Kit to perform RNA isolation / purification Tissue - Rat Brain

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary rat aortic smooth muscle cells

Get tips on using MagMAX™-96 Total RNA Isolation Kit to perform RNA isolation / purification Tissue - rat brain tissue

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary rat marrow stromal cells

Get tips on using AllPrep DNA/RNA Mini Kit to perform RNA isolation / purification Cells - primary rat cortical neurons