We have problems with Trizol RNA preps of purified (negative selection by Miltenyi beads) human CD14+ monocytes. The isolated cells (in the range of 1-5 x 10^6) are hard to pellet (even at high g), subsequent RNA prep w/ Trizol does not yield visible RNA pellets during the precipitation step. I used column-based kits (e.g. RNeasy mini by Qiagen) in the past but would prefer to stick to a quick lysis procedure (we do short-term inhibitor kinetics with the cells, thus have to rely on a speedy protocol) such as provided by Trizol. Is this problem due to the generally low amount of total RNA in resting cells? What other (fast) method(s) and/or combo protocols could we use that avoid RNA precipitation or lengthy lysis and homogenization steps?
Hi Thomas, I have worked with the same kind (human CD14+ monocytes) of cells in the past. I used negative selection by Miltenyi beads to isolate monocytes, in the last step of the protocol, I spun the cell at 300 x g for 10 mins in MACS buffer and got very nice pellet. In order to isolate RNA, I used to save them in RNAlater solution, I extracted total RNA using qiagen RNeasy minikit and the yield was ~50ng/ul from 1X10^6 monocytes, that was good enough to run RTqPCR. later, I figured that I can quickly lyse the cells, using RLT buffer from minikit and store them at -80, untill I am ready to extract RNA. both the procedure were working fine and were consistent. if you are too worried about RNA degradation than use RNAlater solution.
Good question I had problems in my previous lab and could not solve....
Adding RNAse-free glycogen or tRNA (as carriers) to the aqueous phase after TRIZOL and before precipitation should improve your yield. Storing the alcohol-aqueous phase-carrier mix a few hours to O/N at -20°C should also help the precipitation step.
I think that columns are a good solution to this problem. By initially lysing your cells in the Qiagen RLT buffer you have stabilised the mRNA levels, prevented further transcription and inhibited RNAses (by addition of beta-mercapto), especially if you can run them through the shredder columns quickly.
Also as Vincent has pointed out, glycogen is also a good solution to improve TriZol yields, but in my experience keeping the alcohol ppt. step at -20 doesn't do anything to improve yield as most RNA precipitates pretty much immediately anyway.
I have also had excellent results using glycogen without ammonium or sodium acetate that is recommended in some protocols. I also use ethanol to precipitate and I hold it at -20 overnight. I am going after small RNAs though so the overnight precip may not be necessary for larger RNAs.
I agree with Vincent, tRNA should help RNA precipitation. Homogenization is very important,vortex for one minute in Trizol isolation.RNA yield should be the same in resting monocytes. If you use any type of column you will loose all you RNA.
I would agree with all of them but I think Qiagen midi or Fermentas kit could solve the issue.
I do not agree with Lemonica about columns kits. Picopure is good if you have a verry low sample volume. Allpreps DNA/RNA from Qiagen is also verry good for verry small sample. You also can use shredder with both.
Here we store the alcohol-aqueous phase-carrier mix overnigth at -20°C or ~1 hour at -80°C and it helped to improve our RNA yield.
Here is an important point that has not been addressed. Non-specific binding is a huge problem with DNA and RNA. Because of their negative charge, both will bind to positively charged sites on any containers or pipettes used in sample preparation. We use Eppendorf DNA Lo-Bind tubes for all of our DNA and RNA work. We do quantitative analysis with LC and LC-MS and have had good success with this.
Hi Thomas, I have worked with the same kind (human CD14+ monocytes) of cells in the past. I used negative selection by Miltenyi beads to isolate monocytes, in the last step of the protocol, I spun the cell at 300 x g for 10 mins in MACS buffer and got very nice pellet. In order to isolate RNA, I used to save them in RNAlater solution, I extracted total RNA using qiagen RNeasy minikit and the yield was ~50ng/ul from 1X10^6 monocytes, that was good enough to run RTqPCR. later, I figured that I can quickly lyse the cells, using RLT buffer from minikit and store them at -80, untill I am ready to extract RNA. both the procedure were working fine and were consistent. if you are too worried about RNA degradation than use RNAlater solution.
After precipitaion, RNA wont be visible as a pellet in most cases unless it has lot of protein contaminanats. Proceed with the extraction and check with your housekeeping gene.
If you use Trizol Method you can improve your concentration using Glicogen to co-precipitate with RNA.
We used the same method like Hardik Patel (RLT buffer) and it worked fine. The main problem might be that the monocytes don't pellet. Monocytes are usually easy to pellet at 300 to 400g, 10 minutes. Maybe your monocytes got somehow activated (endotoxin contamination of a buffer for example), then they are harder to pellet because they tend to adhere to plastic.
You can try High Pure RNA Isolation Kit from Roche Diagnostics. It is convenient to use without precipitation step.
To the issue of "subsequent RNA prep w/ Trizol does not yield visible RNA pellets during the precipitation step". We work with dendritic cells (5-10 x10 e5) and always use two Glycogen steps, in combination with Trizol (at the beginning and in the precipitation step with isopropanol).
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