I start my primary cell culture in a 24-well plate with 2x10⁶/mL PBMCs obtained through Ficoll gradient. After 24 hours, I wash the wells with saline to leave only adherent monocytes (~1x10⁵) in the plate and add 2mL of complete RPMI medium. On the sixth day of the experiment, I infect half of the wells with L. infantum isolates. After 2 hours, I wash the wells with saline and add another 2 mL of complete RPMI medium to each well. After 72 hours, I remove the supernatant from each well and add 500 uL of Trizol per well for RNA extraction. I combine the contents of two wells in a 1 mL microcentrifuge tube. However, after extraction, I am unable to recover even 10 ng/uL from most of the wells. How could I solve this problem?
Confirm proper adhesion of monocytes to the plate by allowing sufficient time for adherence before adding the medium. Ensure only adherent monocytes are present for RNA extraction.
My assumtion is that you use not enough cells in your assay. Keep in mind that immune cells contain less RNA compared to for example cancer cells. For sufficien RNA yield I typically use at least 1 Mio cells per condition. Good luck!
Bianca Maria Abigail Nyarko to ensure proper cell adhesion, I incubated the plate in CO2 for 24 hours at 37ºC. I tried two methodologies: one method involved detachment (PBS + 5mM EDTA solution) in a 6-well plate, counting monocytes before transformation with re-attachment in a new 24-well plate, and another method without detachment, where the cells were plated in a 6-well plate. In the first method, I had 2.5 x 10⁵ cells per well. In the second method, I initially had 6 x 10⁶ cells per well, but 10% of PBMCs are monocytes, and I observed that only 3% of monocytes adhered to the plate. So, theoretically, with the second method, I had 1.8 x 10⁵ cells per well. In the first method, I lysed the cells with 500 µL of Trizol per well, but in the second method, I used 1 mL of Trizol per well. In the final experiment, I obtained 63.9 ng/µL of RNA with the first method and 162.5 ng/µL of RNA with the second method. These RNAs were eluted with 20 µL of ultra-pure, RNase-free water. I need a minimum of 300 ng/µL of RNA in 40 µL of eluted sample. I am unsure if this is an average concentration of recovered RNA under these conditions or if I can recover more RNA under these conditions.
The average concentration of recovered RNA under the above conditions is 162.5 ng/μL with the second method, which involves no detachment. This method will yield significantly more RNA compared to the first method with detachment (63.9 ng/μL). To obtain the required 300 ng/μL of RNA in 40 μL, you would need to scale up the sample volume by approximately 4-fold, either by using a larger well plate or by pooling multiple well