RNA isolation / purification Cells - immortalized A2780

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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5 years ago

5 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 6 matching solutions for this experiment

Protocol tips
The total RNA was extracted from cultured cells after 24 h incubation with various concentrations of IL-6 or IL-6/IL-8 using a High Pure RNA Isolation kit (Roche, Basel, Switzerland), according to manufacture's protocol. The extracted RNA was purified and diluted in DNase and RNase-free water. The quality and quantity of isolated RNA was measured using a spectrophotometer NanoDrop® (Thermo Fisher Scientific, Inc.).
Protocol tips
Total RNA was isolated from A2780 and A2780CP20 ovarian cancer cell lines with the GenElute Mammalian Total RNA Purification Kit (Sigma-Aldrich). The RNA concentration was read in a nanodrop. RNA quality was verified in a Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). One hundred nanograms of total RNA were used to cDNA synthesis.
Protocol tips
- To yield hign RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
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.


Upstream tips
- Adjust buffers to pH to 6.5 - 7.5 if they have have an acidic pH for better results.
Protocol tips
- An additional wash with 75% ethanol improves 260/280 ratio
Protocol tips
- To capture more amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.
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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