RNA isolation / purification Cells - immortalized HeLa

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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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?

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Protocol tips
the purified mitochondria were resuspended in RNA lysis buffer and incubated at room temperature in column for 5 min to allow binding. The column was washed thrice with washing buffer at 14000 rpm for 1 min. The RNA was eluted in nuclease free water from the column. Purity and concentration of RNA was determined using NanoPhotometer (Implen, Germany). The integrity of mitochondrial and total cellular RNA was checked with Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA).
Protocol tips
after transfection using GenElute™ mammalian total RNA isolation (Sigma), and treated with DNase-1 Amplification Grade (Sigma).
Protocol tips
RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) and converted into 1 µg of cDNA (Thermo Scientific).
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