RNA isolation / purification Cells - immortalized MDA-MB-468

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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Protocol tips
- To yield high 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.

- For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).
Protocol tips
All 4 methods successfully extracted RNA (determined as generating a measurable signal using the Qubit) from as little as one 5 μm section, with geometric mean RNA yields greater than 100 ng (Table 1/Fig. 1). The Qiagen RNeasy FFPE extraction kit gave a superior geometric mean RNA yield of 398 ng
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.
Upstream tips
- Before beginning the ReliaPrep™ RNA Cell Miniprep System protocol, four solutions must be prepared.
Downstream tips
- DNase treament is provided with the kit, but it not required. It is recommended for RT-PCR applications.
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