RNA isolation / purification Cells - primary human endometrial stromal 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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5 years ago

5 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
Total RNA from cultured healthy ESCs and corresponding rhCXCL12-treated healthy ESCs (n=18) was extracted using Qiagen miRNeasy Mini kit (Qiagen GmbH, Hilden, Germany). The quality of the extracted RNA was verified via absorbance measurements at wavelengths of 230, 260 and 280 nm using a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Inc.).
Protocol tips
Total RNA was extracted using an AllPrep Micro Kit (Qiagen, Hilden, Germany). Total RNA samples were subjected to gene expression microarray analysis using a SurePrint G3 Human GE Microarray 8x60K Kit (product no. G4851A, design ID 28004, Agilent Technologies, Santa Clara, CA, USA) by following the manufacturer’s instructions.
Protocol tips
Total RNA was extracted and 1 μg RNA was subjected to cDNA synthesis as described previously [9].
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