RNA isolation / purification Cells - immortalized C3H-10T1/2

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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TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
Following incubation with 200 ng/ml BMP-2 for 1 or 4 days, total RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was also extracted from BMP-2 untreated cells. Total RNA from each sample was quantified using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA)and RNA integrity was assessed using standard denaturing agarose gel electrophoresis
Protocol tips
Total RNA from cultured cells was isolated using RNeasy Plus mini kit (Qiagen) and complementary DNA was synthesized from 1 μg of RNA using a high capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions.
Protocol tips
At the appropriate timepoint(s), total RNA was harvested according to the manufacturer’s instructions (RNeasy Mini Kit; Qiagen, Valencia, CA).
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