I tried several times extrating RNA from human neutrophils and the amount of RNA at the end of the precedure was minimal.
I also tried using colunns from QIAGEN.
Never had any problem with tissue extration or extrations from cell cultured.
Could someone who works with neutrophils share an especific protocol?
Hello Vanderlei
I suggest you use the phenol/chloroform method for RNA isolation. Though it is time consuming it is reliable and you will get a good yield. Also you need to know that to get a good yield the cell number and the time point is very essential. An important point to be noted that neutrophils cannot remain in culture for long. About 80% of human neutrophils become apoptotic when cultured for more than 20 hours. You should work with ice and minimize vortexing steps because RNA from neutrophils is very sensitive.
Regards.
Hi Malcolm, thank you!
In our experiments we keep neutrophils for 4 hours in culture.
I tried various quantities without success.
I know the gene expression in granulocytes are ephemeral, but not like that!
I will try using the method that you suggested.
Machrey Nagel, Nucleospin RNA mini kit works very well for PBMC RNA extraction.
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