RNA isolation / purification Cells - immortalized T98G

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 4 matching solutions for this experiment

mirVana™ miRNA Isolation Kit, with phenol

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation. - To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.	- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA.

Protocol tips
- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation. - To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.
Downstream tips
- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA.

Manufacturer protocol Publication protocol 1 Relevant paper

RNAzol® RT

Molecular Research Center, Inc.

Upstream tips	Protocol tips	Downstream tips
- To reduce RNA degradation do not wash cells before the addition of RNAzol® RT. - To reduce DNA contamination, use sufficient amount of RNAzol® RT based on the area of the culture dish and not on cell number.	To reduce DNA contamination extend the incubation time to 15 minutes before sedimentation.

Upstream tips
- To reduce RNA degradation do not wash cells before the addition of RNAzol® RT. - To reduce DNA contamination, use sufficient amount of RNAzol® RT based on the area of the culture dish and not on cell number.
Protocol tips
To reduce DNA contamination extend the incubation time to 15 minutes before sedimentation.

Manufacturer protocol 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

PicoPure™ RNA Isolation Kit

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
- Can be used when there is a very less amount of starting material.

Upstream tips
- Can be used when there is a very less amount of starting material.

Manufacturer protocol Publication protocol 1 Relevant paper

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