Trying to optimize RNA isolation from 1X10^6 differentiated THP-1 cells by Trizolo. Can any one suggest optimum yield. I could not see any pellet after centrifugation at 13000 rpm for 30 min. What could be reason behind it?
Hello
You said " centrifugation at 13000 rpm for 30 min " ? I think it is after isopropanol precipitation ? Even if you did'nt see any pellet, did you try to hydrate RNA with water and to dose the quantity ? If you had nothing maybe it is due to the previous steps. Did you see DNA (white ring between two phases) after adding chloroform and centrifugation ?
Usually 12500g (for rcf to rpm conversion need to know the radius of the rotor you use) 15min 4C is more than enough to see the RNA pellet from 1 mln THP1 cells (it's a lot!). You can add glycogen to Trizol itself or to aqueous phase just before addition of isopropanol to increase RNA yield and to see air-dried white pellet ( I believe its RNA with glycogen). It's important to add equal volume of 100%isopropanol to aqueous phase for successful RNA precipitation. As already suggested above, you may need to add water after washing and drying of RNA pellet (even if you don't see it) and measure RNA concentration to see where the problem is coming from. You can add Trizol directly to the well after washing of adherent after PMA treatment THP1 cells to avoid massive cells death after scrapping and wait 10 min at RT until cells completely lyse (you can see in microscope that all cells are lysed). For 1 mln THP1 cells I add minimum 1ml of Trizol for good A260/280 ratio.
The use of a colored carrier (ie: glyocoblue or pellet paint pink) will greatly help both collecting and visualizing the RNA pellet.
Best of luck!
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