RNA isolation / purification Cells - immortalized bEnd.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?

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Found 3 matching solutions for this experiment

Aurum™ Total RNA Mini Kit

Bio-Rad Laboratories

Upstream tips	Protocol tips	Downstream tips
- This kit is designed to process upto 2x10^6 cells. - Before using lysis solution, add 500 ul of β-mercaptoethanol (β-ME) to the solution for a final concentration of 1%.	- To perform efficient elution preheat the elution solution at 70C in water bath priorto the elution step. - If there is high genomic DNA contamination, increase DNAse I digestion time and use only the DNAse dilution solution provided in the kit.	- For better downstream application add appropriate volume of 95-100% ethanol to the wash solutions before intial use. - If elute volume is >80ul, there is a possibility of ethanol contamination. To reduce this, add 1-3min of centrifugation time after the final wash step.

Upstream tips
- This kit is designed to process upto 2x10^6 cells. - Before using lysis solution, add 500 ul of β-mercaptoethanol (β-ME) to the solution for a final concentration of 1%.
Protocol tips
- To perform efficient elution preheat the elution solution at 70C in water bath priorto the elution step. - If there is high genomic DNA contamination, increase DNAse I digestion time and use only the DNAse dilution solution provided in the kit.
Downstream tips
- For better downstream application add appropriate volume of 95-100% ethanol to the wash solutions before intial use. - If elute volume is >80ul, there is a possibility of ethanol contamination. To reduce this, add 1-3min of centrifugation time after the final wash step.

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Plus Mini Kit

Qiagen

Upstream tips	Protocol tips	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.

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

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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