RNA isolation / purification Cells - immortalized Calu-3

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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2 years ago

2 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 5 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.
Upstream tips
- This kit can be used for the isolation of RNA from up to 5x10^5 cells, or up to 5 mg of tissue.
Downstream tips
- Always blank the spectrophotometer with your elution buffer.

- When measuring 260 nm absorbance, a unit of 1 on this scale equals to 40 ng of RNA.
Downstream tips
- Include DNAse treatment for 15-20min.

- Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C.

- Use water to elute the RNA that is warmed to ~60`C.
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.
Protocol tips
- For better lysis, pipet the RNA Lysis Buffer over the bottom of the well.
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