RNA isolation / purification Cells - primary rabbit aortic endothelial 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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Found 2 matching solutions for this experiment

TRIzol™ Plus RNA Purification Kit

Thermo Fisher Scientific

Upstream tips
RAECs were incubated in endothelial basal medium, TNF-α conditioned medium (10 ng/ml) or Slit2 conditioned medium (100 ng/ml) in a humidified incubator containing 5% CO2 at 37°C for 48 h.
Protocol tips
Total cellular RNA was extracted using the TRIzol® Plus Purification kit (Thermo Fisher Scientific, Inc.), according to the manufacture's protocol.
Upstream tips
RAEC were used to evaluate NFAT regulation of CSE transcription. Cells were treated with vehicle (ethanol) or CsA (1 µM) for 48 h.
Protocol tips
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies).
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