siRNA / miRNA gene silencing Human PC3 (human prostate cancer cell line)

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

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

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.

Get tips on using Human/Mouse Active Caspase-3 Antibody to perform Western blotting Caspase-3

Get tips on using ScriptSeq Complete Kit (Human/Mouse/Rat) to perform RNA sequencing Mouse - J774

Get tips on using Quantifiler™ Human DNA Quantification Kit to perform DNA quantification Brain tissue

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

Get tips on using PE Mouse Anti-Human CD61 Clone VI-PL2 to perform Flow cytometry Anti-bodies Human - CD61

Get tips on using PE Mouse Anti-Human CD26 Clone M-A261 to perform Flow cytometry Anti-bodies Human - CD26

Get tips on using PE-Cy™7 Mouse Anti-Human CD10 to perform Flow cytometry Anti-bodies Human - CD10