I extracted RNA using the Trizol/chloroform method from primary macrophage that was plated with FBS and P/S.
The 260/280 ratios from nanodrop were as high as 6.5. Some were at 2.6, 2.9, 4.3. Raw 260 were consistently low. And concentrations were in the 100 to 200 range. But I don't even know if I trust it.
It is definitely not because of the extraction method because cells that have not been plated had perfect ratios and concentrations. I have already repeated it already.
Has anyone experienced this when extracting RNA from cultured cells?
Material showing A260/A280 ratio significantly higher than 2 is definitely not nucleic acid.You mention that concentration were “in the 100-200 range”. What is the unit ?
Pierre Béguin pardon my sloppiness. That’s ng/ul. I plan to run the rna through the Agilent bioanalyzer.
My current working theories are that:
1. The oncentrations were too low and that it’s beyond the working concentration of the nanodrop1000. Therefore, the readings were off.
2. I read that FBS has potent RNAse activities and could have destroyed the RNA. I’m slightly against this hypothesis because others have been able to collect RNA from cultured cells with FBS.
With an optical path length of 1 mm (as is the case for the Nanodrop) and a concentration of 100 ng/ul (microliter), the absorbance reading at 260 nm should be 0.2 for DNA and 0.25 for RNA, well within the operational range of the Nanodrop.
If you fear that FBS is the source of the problem, maybe washing the cells with isotonic saline before lysis may help. In addition, if you are looking at total RNA, most of it is actually ribosomal RNA, which is fairly resistant to degradation due to its secondary structure, Even if the RNA prep is badly degraded, it is thus rather surprising that it should be destroyed to the point of no longer yielding any UV-absorbing material.
Hi Theros Ng ,
I have done several extractions of RNA from macrophages and they are certainly a bit more challenging than cell lines. I can tell you that the FBS is certainly not a problem, there must be contamination coming from somewhere else or too much salt. Could you share the protocol you used and any other reagent.
What I normally do to have a pure sample is to use the TRIzol™ Reagent and Phasemaker tubes, and wash 3 times at the end (the washing is crucial for a good quality RNA).
I would also check that your regents are molecular grade as well.
hi Samuel J Lara Reyna
I used 1ml Qiazol, twice 200ul chloroform 600ul isopropanol precipitation in -20c overnight and twice 600ul ethanol washes.
like I said other nonplated samples were perfect.
unless there are more salt in cell culture than nonplated cells. I don’t see the protocol to be the problem.
Hi Theros Ng ,
I would like to highlight that QIAzol is optimised to be used for lysis of fatty tissues and is certainly not Trizol; having said that, I have used it with macrophages and with the correct technique you have as good values as with Trizol.
My main concern is that you are using quite an old method for the extraction. You don't need to add twice 200ul of chloroform or the -20 overnight precipitation. I would suggest you follow the protocol from the manufacturer:
https://www.qiagen.com/gb/resources/resourcedetail?id=6c452080-142a-44a7-a902-9177dea57d7c&lang=en
If you follow this, you should be able to get nice quality RNA.
Samuel J Lara Reyna
I used the multiple chloroform phase extraction to ensure no residual Trizol/qiazol contamination. And overnight precipitation maybe unnecessary but it is to maximize recovery. I have done it enough and got good quality rna for sequencing. it also worked with other primary tissue macrophages in the same set of extraction that has not been cultured.
i don’t think it’s the reason.
thanks for your input.
Update: Ran the sample with bioanalyzer. The RNA quality of those samples with high 260/280 were high. RIN: 9.2-9.8. However, the concentration were low: 11- 28 ng/ul in 10ul volume. Our sequencing requires 500ng of RNA.
For now the solution is to increase number of cells and use carriers such as proteanase K and glycogen to co-precipitate the RNA.
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