RNA isolation / purification Cells - immortalized Neuro2a

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?

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Found 5 matching solutions for this experiment

RNeasy Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- 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

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

Manufacturer protocol Publication protocol 1 Relevant paper

High Pure RNA Isolation Kit

Roche Lifesciences

Upstream tips	Protocol tips	Downstream tips
- To decrease RNase activity,process starting material immeditely or store at 80C. - To increase nucleic yield or purity store all buffer at +15C to +25C.	- To decrease RNase activity,use eluted RNA directly in downstream procedure or store at -80C immediately.

Upstream tips
- To decrease RNase activity,process starting material immeditely or store at 80C. - To increase nucleic yield or purity store all buffer at +15C to +25C.
Protocol tips
- To decrease RNase activity,use eluted RNA directly in downstream procedure or store at -80C immediately.

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Lipid Tissue Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	- If the RNA yield is low, repeat RNA elution, but incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. - All centrifugation steps should be performed at 15–25°C except for phase separation

Protocol tips
- If the RNA yield is low, repeat RNA elution, but incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. - All centrifugation steps should be performed at 15–25°C except for phase separation

Manufacturer protocol Publication protocol 1 Relevant paper

NucleoSpin® RNA

Macherey Nagel

Upstream tips	Protocol tips	Downstream tips
- Aliquot rDNase and store at -20 °C.	- Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.

Upstream tips
- Aliquot rDNase and store at -20 °C.
Protocol tips
- Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.

Publication protocol 1 Relevant paper

TRIzol Reagent

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Manufacturer protocol Publication protocol 1 Relevant paper

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