rna-isolation-purification-tissue-rat-pituitary-gland

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.

Get tips on using Anti-phospho-Histone H2A.X (Ser139) Antibody to perform TissueFAxs phospho-Histone H2A.X (Ser139) - Mouse Human

Get tips on using pCW-LIC to perform Protein expression and purification Bacteria - Escherichia coli Fbxo7

Get tips on using exoEasy Maxi Kit (20) to perform Purification of extracellular vesicles Exosomes - Plasma

Get tips on using exoRNeasy Midi Kit (50) to perform Purification of extracellular vesicles Exosomes - Plasma

Get tips on using IMPACT™ Kit to perform Protein expression and purification Bacteria - Escherichia coli GmrSD

Get tips on using pGS-21a Vector to perform Protein expression and purification Bacteria - Escherichia coli p19

Get tips on using Ni-NTA Agarose to perform Protein expression and purification Insect cells - Hi5 TYR

Get tips on using QIAexpress Type IV Kit to perform Protein tag Purification of His-tagged proteins

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.