RNA isolation / purification Cells - primary rat cardiac fibroblasts

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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Protocol tips
The following might help to increase RNA yield

- Heat the DEPC Water to 70°C before adding to the column. - Increase the incubation time to 5 minutes.

- Increase the elution volume. - Repeat the elution step with fresh DEPC Water (this may increase the yield, but decrease the concentration).

- Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).
Protocol tips
- Check your lysate, the viscosity should be low, high viscosity can result in low RNA yield and high DNA contamination. Perform extra vortexing, pipetting or add more lysis buffer.

- Warm the elution buffer to 60`C for higher RNA yields.
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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