Hello all,
I am trying to isolate RNA from neuronal cells using Trizol ,chloroform, isopropanol. After reading the OD, the concentration of RNA is good, but their quality is not good.
So, I started purifying the RNA by Qiagen's RNeasy kit to remove contamination. But unfortunately, after doing the procedure, the concentration of RNA decreased significantly and I lost RNA.
Has anyone done this experience before?
Thanks in advance
A few things regarding all the steps you mentioned.
1. You can get good quality RNA from trizol preps. I am unsure how much cell material you are processing neuron cells (I mostly use trizol for bacteria), but less is much better. Too much cell material can definitely cause poor RNA quality.
2. When you take the upper phase, make sure you leave some upper phase behind. You risk taking the middle and lower layers when you separate out the rna.
3. I prefer to use ethanol than isopropanol for rna precipitations from trizol. isopropanol precipitates salts, thus making your rna pellets saltier and requires more washes prior to resuspension.
4. I typically wash my rna pellets 3 times with cold 80% ethanol
5. Columns are great for cleaning up RNA, but make sure you are following the manufacturer's directions for loading RNA and elutions. Also, running rna through columns does result in rna loss due to insufficient elution and/or loading issues. From columns, I typically elute 3 times with warm water or elution buffer. If you are concerned about eluting all rna off column, just elute in a large volume and precipitate using ethanol plus sodium acetate and glycogen
6. have you looked into using the direct-zol rna isolation kit from zymo? I love using this kit for RNA isolation from cells. It is worth it and you get great quality rna each time.
What about the integrity? I'm pretty sure that at least for me, the solution, was to was to heat the TRIzol to 56C before adding it to the pulverized and frozen tissue. Another key step is to discard a portion of the interphase in after the first centrifugation. I'm pretty sure that using the columns to clean the RNA would decrease the yield, maybe incorporating the steps I mention above would result in a more decent RNA quality and integrity without compromising the yield.
Good luck!
Hello Elham Amini. E.A
If you already have a proper RNA concentration and the problem is with the quality, I would still recommend going with the TRIzol method. Try that method again and be very cautious about pipetting, especially when you take the upper aqueous layer. It's okay to leave some behind rather than accidentally disturbing the interphase or the organic layer.
RNA cleanup kits are also efficient if you still struggling with a significant amount of contaminants in your sample. But as you experienced, it does lower the yield. Carefully try the TRIzol method once again.
Best wishes!
Dear Kyle Skalenko, thank you so much for your reply. Actually, I extracted the RNA form neuronal cells, by Trizol,....., however the RNA concentration was good, the quality was low. So, followed the exact protocol, step by step for RNA cleanup by Qiagen's RNeasy kit. At the end, I saw that the RNA concentration became so low, and in some samples I lost the RNA. In the protocol it mentioned that RNA is eluted in 30–50 µl water.
Based on your experience, you mean it is better to use absolute ethanol instead of isopropanol for RNA precipitations ?
How much sodium acetate and glycogen are needed to add to ethanol?
Dear Erik Diaz, thank you for your reply. I have not check integrity, only concentration and quality 260/280. I found that the quality of RNA was very low, then used the RNA cleanup. Unfortunately, after cleanup by kit and column, I lost my RNA.
So, based on your comment, you mean it's better to heat the TRIzol to 56C before adding to the cells?
For adding the Trizol to the cell for RNA extraction, it's better to add the Trizol directly to the flask or plate of cells or it's better to harvest cells by Trypsin, then count them, then add Trizol to the pellet of cells? Which way is better?
Dear Sandali Geethma, thank you for your comment. Yes, you are right. I lost the RNA completely after RNA cleanup, however before this process the concentration was good and high only the quality was low and got contamination.
Ok, sure, will try Trizol again, care about the separation part.
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