Hi all,
I am having some issues with my Trizol-based RNA isolations. I am attempting to harvest RNA from bone marrow derived macrophages (3x10^6). After the initial separation with chloroform I observe the characteristic three phases. I collect the aqueous phase and perform a second chloroform wash (been advised that this helps remove extra phenol). After centrifugation, three phases are again observed (top and bottom are clear with a pinkish interphase). I collect the top most layer and continue processing according to the protocol. I then add isopropanol and 10 ug of glycogen. After spinning oddly I observe another phase separation. I cannot see an RNA pellet but instead observe a white cloudy suspension in the interphase where I fear my RNA is. I discard the supernatant and continue with ethanol washing where I think my pellet should be. Unfortunately, my RNA yield was very low (5-9 ng/uL).
I have saved the supernatant but am unsure how to collect my RNA (in the event that it is still there).
Thanks!
Hi there, after adding isopropanol, you should not see another phase separation. I assume strongly that you are touching the middle layer (interphase) some way which must be avoided. Usually we collect the top aqueous layer leaving 10-15 microliter at the top of interphase. Thus you get a clear supernatant. Hope this helps.
Hi Sharmin,
It was not a clear phase separation as my question indicated. I did notice that after adding isopropanol the solution turned milk-white almost. Unfortunately, nothing precipitated out of solution even in the presence of 10 ug of glycogen. Is there a way that I can use the solution that I have left-over (isopropanol+aqueous phase from second chloroform wash) to harvest any RNA? Could I run the sample through a spin column?
i've never done a second chloroform wash, or use glycogen for precipitation, and always get enough RNA for RT-PCR studies. If this is the way they've always done it in your lab it must work out OK. You should try the protocol as is stated in the datasheet.
I work with Stem Cells also from bone marrow.
I guess you already have a protocol from the manufacturer of TRIzol reagent , nevertheless I attached for you one I use to compare. You will see that along the process you can save some steps of extractions for further purifications. I must say I have never used glycogen in TRIzol RNA extraction and I used TRIzol regaent from different manufacturers.
Best of luck.
Hello Sushnath,
If I were you, I would re-do the experiment from the beginning. Something must be wrong what I understand. I am using Trizol and also never used Glycogen, still getting a decent amount of RNA. If you re-do the experiment, kindly follow my previous tips. Best wishes :)
Hi,
So brief update. I went ahead and looked over my discarded supernatant (aqueous phase from 2nd wash + isopropanol) which had been sitting in 4 C for 48 hrs and was able to precipitate a white pellet for all the samples. Not sure why I was unable to do this during my first attempt. I dissolved the pellets in RNAse-free water (20 uL). The samples had yields ranging from 500-1200 ng/uL.
A260/280 = 1.8-2.0
A260/230 = 0.5-1.4
I am worried that the low A260/230 is due to degraded RNA. Should I try to do another precipitation to further clean the pellets or will the samples have degraded too much in isopropanol solution to even bother?
Most of the time you won't get good ratios after this kind of RNA isolation. Especially if the slighest trace of Phenol or Ethanol is still left in the sample. If spectrometric measurments are really important, we alwas to a column based clean-up (keep in mind that you need different sep-ups depending on the size of the RNA you want to analyze). The Phenol-Chloroform´isolation method with the RNA precipitation gives the highest total yields including small and long RNAs (in our hands) and we make the additional clean-up step for mRNA arrays.
Spectrometric measurments won't tell you the quality of your RNA. For a standarized and quantified RNA integrity number (RIN) you would need to run an Agilent RNA analysis. If you just want to see whether it is degraded or not, just run a 1% agarose gel.
For your amount of cells I would also skip the glycogen, this is usually added if you want to isolate small RNA amounts to help with the precipitation as well as the second chloroform wash.
Hi,
I once had a student who got two phases after adding isopropanol (IPA) to the water phase and spinning. The reason for that was that he simply didn't mix the tube after adding isopropanol. I know it's trivial, but just wanted to check whether you are making sure that you do a proper mix after adding isopropanol - give it a good shake or vortex.
Some extra info: the solution can turn milky white after adding IPA because the water phase contains a lot of salts which precipitate in isopropanol. After you mix the tube thoroughly, it is no longer a mix of water and isopropanol, but an actual 50% isopropanol solution in which salts should be perfectly soluble and therefore the solution should turn transparent again.
Hi,
I use Trizol and never used Glycogen or two wahes with chloroform. But I had problems with A260/230 and it was problably contamination with salt and protein. I did a second wash with 100% isopropanol and than 75% ethanol. It was very good but you will loose some RNA in this process. I hope this help ; )
Hello Mr. Kumar
I fully agree with madam Fabian, Sometimes the errors in the nanodrop reading are very common due to alcohol contamination and also because of froth formation. Moreover, nano-drop readings never can be considered as a chronometric test. The overall quality of an RNA preparation can be assessed by electrophoresis technology on an agarose gel; this would be enough to give the information about RNA yield as well. While running the gel make sure you run a positive control on the gel so that you can do the troubleshooting of problem leading to unusual results. Because if you want to do further gene expression analysis, it would be very important to check your RNA quality, otherwise you may end up with a bad cDNA synthesis which will ultimately lead to a bad analysis for RT-PCR.
I guess, the below-mentioned link will help you with your concern.
https://www.thermofisher.com/kr/ko/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/agarose-gel-electrophoresis-of-rna.html
I hope it will help you.
Hi
You might find your answer here.
https://www.neb.com/tools-and-resources/troubleshooting%20guides/Troubleshooting-Guide-for-Total-RNA-Extraction-and-Purification
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