RNA isolation / purification Cells - immortalized PC-3

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

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. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

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).

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

TRI Reagent® Sigma

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
- 1-Bromo-3-chloropropane is less toxic than chloroform and its use for phase separation decreases the possibility of contaminating RNA with DNA. - The chloroform used for phase separation should not contain isoamyl alcohol or other additives.

Upstream tips
- 1-Bromo-3-chloropropane is less toxic than chloroform and its use for phase separation decreases the possibility of contaminating RNA with DNA. - The chloroform used for phase separation should not contain isoamyl alcohol or other additives.

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	RNA was isolated from independent preparations of the indicated cell type using the RNeasy mini prep kit (Qiagen) and quantitative real-time RT-PCR (qRT-PCR)

Protocol tips
RNA was isolated from independent preparations of the indicated cell type using the RNeasy mini prep kit (Qiagen) and quantitative real-time RT-PCR (qRT-PCR)

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

Direct-zol RNA Kits

Zymo Research

Upstream tips	Protocol tips	Downstream tips
- Highly effective when processing various sample types in TRI Reagent®, TRIzol®

Upstream tips
- Highly effective when processing various sample types in TRI Reagent®, TRIzol®

Manufacturer protocol Publication protocol 1 Relevant paper

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