RNA isolation / purification Cells Cancer cell lines

The RNA interference (RNAi) is used to inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA) play a central role in RNAi. Few points have to considered to increase the transfection efficiency of siRNA. Always use healthy, actively dividing cells to maximize transfection efficiency. The confluency of cells should be between 50-70%. Always use the most appropriate siRNA concentration to avoid off-target effects and unwanted toxic side effects. Positive and negative controls should be used for each and every experiment to determine transfection efficiency.

Get tips on using PrimaPure Porcine Endothelial Cells Growth Medium to perform Mammalian cell culture media PAOEC

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

Get tips on using LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation to perform Live / Dead assay mammalian cells - Spleen 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.

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 RIPA Buffer to perform Protein isolation Mammalian cells - 3T3-L1

Get tips on using RIPA Buffer to perform Protein isolation Mammalian cells - THP-1

Get tips on using Trichloroacetic acid to perform Protein isolation Mammalian cells - THP-1

Get tips on using RIPA Buffer to perform Protein isolation Mammalian cells - CHO-K1