RNA isolation / purification Cells - immortalized HEK 293

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.

Start discussion

Found 1 discussion for this experiment

Discussion

1 year ago

1 year ago by Ralf Friedmann Switzerland

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

Share your thoughts or question with experts in your field by adding a discussion!

Found 3 matching solutions for this experiment

Protocol tips
Total RNA was isolated from HEK293 cells and 2BS cells using an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. The quality of the RNA samples was examined by quantifying the A260:A280 ratio (the minimal acceptable ratio is 1.7) and the 28S/18S by visualizing rRNA bands in agarose gel (the minimal acceptable ratio is 1.5).
NucleoSpin® RNA

Macherey Nagel

Protocol tips
After pre-incubation with or without MAPK inhibitors followed by stimulation with or without 10 µM HA (as indicated) for 4 h, the medium was removed and the cells were washed twice with PBS. Total RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol.
Protocol tips
Total cellular RNA was isolated using an RNA isolation kit (RNA Stat-60, amsbio, Abingdon, UK)
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!
Become shareholder Discussions About us Contact Privacy Terms