ChIP H3K27me3 Rat Mouse

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.

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 TRIzol Reagent to perform RNA isolation / purification Tissue - Human Retina

Get tips on using LAMP-1 Antibody (H4A3) to perform Autophagy assay cell type - Proximal tubular cells (rPT)

Get tips on using AllPrep DNA/RNA Mini Kit to perform RNA isolation / purification Tissue - Human Retina

Get tips on using Gibco DMEM/F-12, HEPES to perform 3D Cell Culture Media hiPSC-derived retinal organoids

Get tips on using Anti-Beclin 1 antibody (ab62557) to perform Autophagy assay cell type - Proximal tubular cells (rPT)

Get tips on using DMEM, high glucose, GlutaMAX™ Supplement, pyruvate to perform 3D Cell Culture Media hiPSC-derived retinal organoids

Get tips on using Gibco™ DMEM/F-12, GlutaMAX™ supplement to perform 3D Cell Culture Media hiPSC-derived retinal organoids

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.