Hello everyone,
I am currently trying to set up at a new lab in order to begin experiments on osteoclasts. Before starting anything, I wanted to make sure that BMMs have indeed differentiated into osteoclasts by using TRAP staining and PCR.
In our lab, we harvest bone marrow-derived macrophages (BMM) from the tibia of 5 week-old female ICR mice for osteoclast differentiation. For BMM differentiation, I seeded 2 x 10^5 cells per well with full alpha MEM and M-CSF (30ng/ml). I used 3 6-well plates in order to retrieve cells on Day0, Day2, and Day4 of differentiation. On the next day, I retrieved the Day0 plate using 1ml of Tri-RNA reagent and changed the media (containing M-CSF and RANKLE (1:1000)) for the other two plates. I retrieved the rest of the plates on appropriate days.
After retrieving all cells, I performed RNA isolation followed by RNA quantification (ND-1000), reverse transcription, PCR, and gel electrophoresis. My problem here is that I'm getting nothing on gel for Day0 with actin, GAPDH, and HPRT primers. I triple-checked all my steps for gel, PCR, and reverse transcription using other cells and the technique does not seem to be the problem. I performed RNA and DNA quantification using Nd-1000 (I know they are not super accurate) and I've attached the results as image files.
Please help me figure out what made the Day0 bands disappear! Thank you in advance:)
As you said nanodrop is not accurate. So i recommend you to run your RNA on a gel to check whether it is intact.
You also haven't listed your 260/230 ratios for the RNA. Trizol/Tri-reagent based RNA isolations use guanidium salts, and these have a tendency to carry over into the final RNA (leading to low 260/230 ratios). As chaotropic salts, these will inhibit cDNA synthesis and PCR considerably.
If your Day0 RNA is really salty, you may simply have failed to reverse transcribe any of it.
Determine the 260/230 ratios for your RNA, and (in my opinion) reclean (usually just precipitating the RNA again suffices) any that show ratios lower than 1.70.
RNA that is intact and RNA that is degraded will give you the same values on a Nanodrop readout. I strongly recommend you run an RNA gel (as Dulshara recommended) to see that you have intact ribosomal RNA bands. This isn't a perfect indiciation that your target mRNA species are intact, of course, but it at least is an indication of the overall integrity of your RNA preparation.
How many hours after plated you have harvested your cells for assays on day 0 ?
Whether your cells were healthy on day 0 ? may be the genes were not expressed.
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