RNA isolation / purification Cells - primary human epithelial 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.

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4 years ago

4 years 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?

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Protocol tips
In order to monitor the response of Dex the transfected cells, we also monitored AQP5 and GAPDH expression at the mRNA level: total RNA was extracted from a 100 μL trypsinized EpH4 cell suspension with the Quick-RNA MiniPrep Kit (Zymo Research). All steps were performed according to manufacturer's instructions. Expression changes were monitored as described above (Extraction of mRNA and analysis with qPCR), except that the SensiMix SYBR No-ROX One-step Kit (Bioline) was used for onestep qPCR.
Protocol tips
Three transwells from each experimental condition were harvested and pooled to isolate RNA by using the RNAqueous kit for total RNA purification from Ambion–Applied Biosystems (Austin, Tex). RNA concentration and integrity were determined by using the Agilent 2100 Bioanalyzer system and Agilent RNA 6000 Nano Chips (Agilent Technologies, Foster City, Calif).
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