I am isolating RNA from human neutrophils with the goal of performing RNA-Seq to assess the transcriptome under various conditions.
I have been isolating neutrophils from human blood using the Miltenyi Maxpress whole blood neutrophil isolation kit. I lyse the neutrophils (apx 10 million cells) in RLT buffer with 1% 2-BME and I pipette vigorously to lyse the cells and them store in -80 overnight. The next day I extract RNA using the Quaigen RNeasy kit, and while the quality has (sometimes) been ok, my yield is too low (1000ng if I am lucky)
Does anyone have any recommendations for how to improve my yield?
I have tried the Trizol-Chloroform method as well, and the resulting RNA was unusable (260/280 of 1.4).
Hello! We like to use QIAgen RNeasy columns with DNAse and TRIzol reagent. The protocol is in the appendix of the RNeasy micro manual. It's nice because you get a chemical extraction, but then you precipitate on the column and can clean off genomic DNA with DNase. So, if you have a very small number of cells, and your not getting any RNA using the RNeasy micro kit, then using TRIzol (phenol/chloroform) with a plugged capillary tube carefully mounted in a microcentrifuge tube, with a syringe needle, to get the maximum aqueous layer and then very careful precipitation with the addition of super clean (ambion) glycogen, will most likely be necessary. This process is aided by the use of a speed-vac. Good luck!!
I just re-read your question the TRIzol RNeasy hybrid should work awesome. In the Molecular Cloning manuals there is a prep for LiCl precipitation of total RNA. If you have access to a speed-vac you will really be able to clean up the total RNA. With TRIzol it will denatured all the RNases and proteins so it is good to do as soon as possible in your isolation. You should get very high RIN.
I agree with Matthew, I used TRIzol RNeasy hybrid for neutrophil and it gave me good RNA quality, but i would use more neutrophil if you could
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