I am trying to determine what best time-point to harvest my cells, HMVEC, after transduction with long non-coding RNA to determine changes in mRNA levels, specifically VEGFR2/KDR.
I am capable of achieving ~90% transduction efficiency by assessing GFP+ cells via fluorescent microscopy as my adenovirus is tagged with GFP. My current protocol is:
Day 0: Seed cells
Day 1: transduce cells with adenovirus (24 hours post seeding)
Day 2: change media (24 hours post transduction)
Day 3: harvest cells
What would be an ideal time point to harvest cells to determine changes in mRNA levels? I was thinking between 16-24 hours post transduction. Would appreciate any input or advice, thanks in advance!
Hi Mark,
Since you can achieve ~90% transduction efficiency according to GFP+ cell assessment, I would recommend you to detect mRNA expression of your genes of interest at different time point (12 h, 16 h and 24 h, with triplicates per time point). There you might know at which time point it's optimal.
In our previous studies, we infected human SMC instead of HMVEC with adenovirus (MOI=20), normally after 24 hr of infection we changed fresh medium and treated the cells with vehicles or drugs. 6 h or 12 h after drug treatment we collected cells pellets for RNA isolation and further mRNA expression in response to drug treatment or RNA sequencing.
Hope it works for your experiment.
Best
Dear Mark,
I also recommend different time points. Note that you need to learn about transcription rates and mRNA turnover of the target of interest in your cells. Maybe look at https://www.researchgate.net/publication/24021520_Down-regulation_of_Vascular_Endothelial_Growth_Factor_Receptor_2_Is_a_Major_Molecular_Determinant_of_Proteasome_Inhibitor-Mediated_Antiangiogenic_Action_in_Endothelial_Cells
and other publications. You expect up or downregulation? Could make a difference.
Best Boris
- Badges
- Science method