I am trying to isolate RNA from monocytes, but using the Macherey-Nagel Kit (NucleoSpin RNA Plus) I only seem to get concentrations of 10-20 ng/µL RNA in 20 µL elution volume, which is barely sufficient for my follow-up experiments.
I culture 750,000 monocytes in 500 µL RPMI 1640 medium, supplemented with GlutaMAX, NaP, gentamicin and human serum for 24 hours at 37 oC. Then I harvest them by adding LBP lysis buffer to the cells (and resuspending thoroughly) -> freezing them in liquid nitrogen and then storing at -80 oC until subjected to RNA isolation.
How can I get a higher RNA concentration?
Please have a look into this discussion page:
https://www.researchgate.net/post/How_to_improve_RNA_yields_from_resting_human_monocytes
When I am getting low RNA yield I try rinsing/washing the well with more buffer to get the cells may be stuck behind. So add your lysis buffer and incubate then remove. add more buffer to the well and combine with the first. Another thing that will help is ensuring you have good lysis and are protecting RNA one cells are lysed. What is in your lysis buffer? how long do you let the lysis go on? if you are freezing it right away then thawing and immediately isolating you may have insufficient lysis. Also, add some RNase Inhibitor to the lysis buffer to help protect your RNA. Also, when you elute from the column you can get more if you reload the eluate and spin it again, I have seen this increase yield. Or when you add the water/elution buffer if you let it sit for 1-2 minutes to fully saturate then spin that can help too.
I perform RNA extraction from cells using trizol.
I have used kits with columns, but the amount of RNA is always very low, I recommend using Trizol. Add trizol directly to the culture dish once the medium has been removed. And leave precipitating with isopropanol overnight at -20°C.
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