RNA isolation / purification Cells - immortalized NK-92

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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1 year ago

1 year 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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Upstream tips
- Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample.

- Different samples require different methods to achieve complete disruption.

- Incomplete disruption results in significantly reduced nucleic acid yields.

- Overloading the spin columns significantly reduces nucleic acid yields.
Protocol tips
- Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption.
Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids.
Excessive homogenization, on the other hand, results in shorter genomic DNA fragments.

- The centrifugation temperature should be 20–25ºC.

- Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.
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.
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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