RNA isolation / purification Cells - immortalized C6/36

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
Total RNA from C6/36 cells at rest was isolated with RNAeasy (Qiagen). Total RNA from cells at rest was enriched for messenger RNA with oligo dT dynabeads (Invitrogen, Carlsbad, CA, USA). Total RNA from mock-infected and Zika-infected samples was subjected to SISPA multiplex library construction [53] and sequenced by Illumina NextSeq 1 × 150-bp sequencing.
Protocol tips
Detached cells were transferred into a sterile 2 ml tube and were stored immediately at −80°C until total RNA extraction. Cells were homogenized in 350 µl RLT buffer in QIAshredder spin columns (Qiagen, Hilden, Germany) prior to total RNA extraction with Qiagen RNeasy Protect cell mini kit (Qiagen) according to manufacturer's instructions.
TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
Total RNA was isolated by using TRI-Reagent (Molecular Research Centre) at different time points after infection, and enriched for small RNAs by using PureLink miRNA isolation kit (Invitrogen).
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