I have cells at near confluence which I plan to harvest soon. A density of 2.0x10power5 is needed for RNA harvest. What amount is suitable for the assay (RNA harvest) at that density? Secondly, how do I ensure that harvest all cells after dislodging?
Hi Stephen..Plz make your question clear and also mention what type of cells you are using? Plz also explain how do you harvest your cells because different people follow different ways for cells harvesting.
Thanks Muhammad...i am using SCC 25 cells from ATCC. I plan to harvest by trypsinization for dislodging, the addition of media to deactivate trypsin, centrifuge and store the pellet in liquid nitrogen fro molecular assay.
Dear Stephen,
I use work with 1x10^6 cells/well in six well plate, treat cells for six hours and collect them in 1 ml of trizol and keep it in -20 till further use.
Sagar......thanks. Please elaborate how the procedure was carried out. If i harvest and put in liquid nitrogen immediately, will i achieve the same effect? will appreciate your answer as soon as you can
Trypsinization followed by deactivation and washing the culture dish with the same media will perfectly work well. You can easily check the adherent cells (if any) through microscope. Centrifugation followed by washing with PBS is highly recommended. I would suggest you to dissolve th pellet in Trizol and process it directly or store it at -70C for later use rather than storing the pellet. For RNA preparation you can harvest your cells directly with trizol as Sagar rightly explained. To this end you remove the culture media and wash the cells with 1X PBS (2-3 times) followed by the addition of Trizol directly to the culture dish. Kepp it at room temeperature for short time (along with gentle rocking or pipetting to dislodge and dissolve the cells) and then process it as described previously. 1x10^6 cells per sample are suffcinet enough to give you a good amount of RNA pellet. For cells to Trizil ratio please check your kit instructions. Good luck.
I would caution against trypsinization, depending on which gene(s) your interested in you may alter RNA expression profiles of some highly labile or short-lived RNA species (FYI-trypsin can itself act as a signal because it activates PAR signalling). Your best bet is to solubilize and stabilize the RNA directly in the dish/flask. Trizol is a good affordable option for this. If you do want to remove the cells and store a pellet for later processing I suggest using cell lifters/scrapers on ice.
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