RNA isolation / purification Cells - immortalized KCL-22

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

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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 3 matching solutions for this experiment

Protocol tips
- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C.

- Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation.

- To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.
Downstream tips
- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA.
Protocol tips
All FFPE sections were deparaffinised using an automated protocol on the Leica autostainer XL, involving immersion into xylene twice followed by immersion into 100% ethanol twice then left to air dry for 15 min, with the exception of the two Promega kits where no deparaffinisation was performed. All extraction kits were used according to the manufacturer’s instructions including elution volumes quoted and all optional DNase 1 steps in RNA extractions.
TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.
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