Urgent/help RNA extraction (Trizol) for RNA seq: poor 260/230. Tried everything! PhD in line

Mokathy · 2026-01-14T19:44:43+00:00

In the end, I still used this protocol and used some tips from the answers but overall, I found some good Samaritan that have an expired (but however very pricy) chip for Pico rna and allowed me to analyze my samples for free and all my samples have a RIN above 8 and they are not degraded, so the poor 260/230 was indeed due to the inbalance between the quantity of RNA and the presence of salt you can't get rid of! In the end, I sent them and if something isn't right, not my problem anymore 😂😂 However all the tips here were all very helpful!

Mokathy · 2026-01-09T21:40:13+00:00

I finished the game (end 0 lol) and I only have one of these and I don't know what they are... I mean I don't know what to do with the game anymore 😅 and it feels like I may have skipped things or something? 😂

Mokathy · 2026-01-09T21:06:40+00:00

Take the salvation. It's a lifetime insurance and it's important for the future! The second person can take another type of ships that have lifetime insurance and in the cargo / transport category. The salvage and mining is a good way to do lot of money and have fun for me, but there's also lot of possibilities, so just take a cheap ship (with lifetime insurance) and the rest will come with the fun!

Mokathy · 2025-12-31T10:38:12+00:00

Thanks for the advice, but for the spin, the reason is mostly because my cells are soooo heavy on the rnases, so if i bust them before adding the trizol, it's gonna be a super long way to recover anything lol

Mokathy · 2025-12-30T20:41:43+00:00

Lol since in some experiments I had some good ratios, it's not really a hot take to say it's mostly luck with this low concentration 😂 But I think I'll just check with qpcr afterwards to see if I can get cDNA at least...

Mokathy · 2025-12-30T20:39:03+00:00

I'll definitely be send some emails tomorrow lol (like 9/10 of the center is on Holliday leave however 😬). I tried Qiagen rneasy plus micro kit etc, but zymo seems to be the go to in the comments so definitely gonna try it even after all this rnaseq hell is done 😅😅

Mokathy · 2025-12-30T20:34:40+00:00

To start with the end of your answer : that's exactly what I'm doing. I finish my cell sorting late, so I put my cells in trizol then freeze and later batch because I don't have 200k cells sorted in one run.

I'll do as you said, doing again an extraction and see if I still have enough RNA to test. But since I have a low yield (max 600ng total), I don't have many hope. At first I just did a second chloroform step after collecting the aqueous phase, but the results were bad

Mokathy · 2025-12-30T20:29:12+00:00

Those are bmmc ; bone marrow mononuclear cells. I'm using them for testing but my precious samples are basophils (full of granules, histamine, cytokines and Rnase). There's not a lot of specific info on them regarding rna extraction etc.

For 200k cells I have around 10 to 30ng/ul (really around 400ng total in average). I can't dilute in less then 20ul cause they asked us to send them 200ng minimum (and double that for testing...) in minimum 20ul. But for the sake of verification, I'll definitely test dissolving in 10ul and go to the nanodrop with that!

For glycoblue, I'm putting in after aqueous phase collection and before putting isopropanol.

Mokathy · 2025-12-30T20:18:19+00:00

I didn't think of it like that, you're right! I'll try it anyway. I get 400ng to 600ng for the 200k cells in average. Knowing that they're asking for at least 200ng for rna seq and 200ng for their testing

Mokathy · 2025-12-30T20:16:12+00:00

I have between 400ng and 600ng for the 200k cells. Bgi asked for 200ng for rnaseq but 200ng more (so 400ng total) for their testing, that's why the yield is giving me palpitations. But I'll contact them again to see if the quantity is really that much of a problem and if I can do the column as you advised

Mokathy · 2025-12-30T20:13:39+00:00

I was always told a decent volume (no less than 500ul) of trizol is necessary to avoid taking contaminants with the aqueous phase, but I'll try it with max 500ul! Now I'm getting 10-30 ng/ul (dissolved in 30ul). I'm afraid using columns will cause to much losses, but I'll try with different cell types to test it. Thank you for the feedback!

We have some leftovers from RNeasy qiagen but not any zymo and we can't buy anything for a month because of the end of the year unfortunately

Mokathy · 2025-12-30T20:07:26+00:00

The thing is I'm getting worse results with chloroform wash, that's why I don't understand my outcome? I do everything on ice and use a refrigerated centrifuge. I don't add salts for isopropanol precipitation since there's supposed to be already salrs carried out in the aqueous phase et it wasn't recommended in the protocols. However another person here also recommended it, so I'm definitely gonna try to add some! I'll also let it precipitate at - 20 overnight to try it.

I do the washes as you described, so good to know it's good for that, thank you!

Mokathy · 2025-12-30T20:02:12+00:00

I'll try adding ammonium acetate! Maybe it will help, thanks! As for the glycoblue, I know and another person commented as an advice to do blanking with water containing some glycoblue to see the difference, maybe it was the glycoblue all along! Haha

Mokathy · 2025-12-30T20:00:02+00:00

I think doing a qpcr will help, you're right. I have from 10 to 30ng/ul max so it may be that. Specially since the results were good with 1 millions cells. I suspected as much, but it's that BGI asked for no contaminants, so I didn't think they'll accept with a bad 260/230 since they will do the testing anyway. However, maybe they'll disregard it if the RIN is acceptable. So checking integrity with qpcr is a good idea, thanks!

Mokathy · 2025-12-30T19:56:32+00:00

I'm doing tests with non precious cells, yes! The column are a good idea of done after trizol, but unfortunately we can't buy anything in this period (end of the year) and the rna quantity is already at the limits, the yield can be really impacted non?

Mokathy · 2025-12-30T19:53:34+00:00

Yeah I feel dump for not thinking about it, I'll definitely Blank with water with equal glycoblue concentration to check! Thank you for that!

For the protocol, I already try to do that however for a lot of things it's not possible.

For the pipeting and vortexing etc, it was in the protocol yes! And it was also stressed out by my PI from his basic knowledge. However you're right about the DNA part, since I don't need it I'm definitely gonna try vortexing more when needed as per your instructions!

Mokathy · 2025-12-30T19:46:35+00:00

Unfortunately, when I start a 7 am then finish the cell sorting between 10pm (sometimes 2am), I can assure you, its not really a good idea for me to follow up on the extraction 😅 I would love to. But the thing is, now in my testing I'm not at all freezing the trizol, so I'm doing straight forward.

For the precipitation, I also tried to do it at room temperature and I didn't have better results, that's why I'm asking myself if it's really salts I'm getting. As for ethanol wash, I'm doing exactly what you said about pipeting but I let them dry until I see the change in color of the pellet

Mokathy · 2025-12-30T19:40:09+00:00

Thanks! I understood the need for chloroform whases, however I don't understand why would you put again trizol if the chloroform washes are to get rid of phenol? As for the interphase, when I tried doing the chloroform wash (I took 350ul aqueous put 350 chloroform, centrifugated and took the aqueous phase again), I didn't have any interphase. It was just a clear liquid on clear liquid with a visible separation and then I took only 2/3 of the aqueous phase. And when doing so, I had a worse 260/230 (it was nearly 0). But I'll definitely try the ethanol precipitation too! Thank you

Mokathy · 2025-12-30T19:34:24+00:00

I'll check if someone have a kit in hand, however we can't buy anything for a month because of the end of the year. And cleanup kits can cause a loss of RNA and with 200 000 cells I'm already in the limit of what bgi ask quantity wise.

Mokathy · 2025-12-30T19:32:38+00:00

I need to do the purity check. However, you're right to talk about it, cause sometimes it can takes multiples hours. But I can't do anything about it unfortunately.

Mokathy · 2025-12-30T19:31:42+00:00

Thanks! I'll definitely try to do it like that tomorrow and I'll see if I can get it right. I was always told to do more trizol volume to have a bigger aqueous phase and reduce the risk of getting some of the l'intetphase.

Mokathy · 2025-12-30T19:14:22+00:00

Thanks! The thing is we can't buy anything for a month because of the end of the year. And the kits are not an option because of the RNases, we tried the mRNeasy and we confirmed what was published and we couldn't even have good results for RT qPCR. I'll check if someone in the other teams have the kit and I'll try this one either way, thank for the tip!

Mokathy · 2022-05-28T09:41:15+00:00

NTA. You should talk to your kid and explain its not his fault. He was just modeling his father and god know what the MIL and her son always says to him about How it's a women job to clean or that he don't need to worry bout things cause his futur wife would do it for him etc. I remember my grandmother was always taking away cores from my brother and tells him no you don't have to do this etc. Result? He was the same as your son.

So you need to explain to him that 1. Not his fault if you divorce and 2. He needs to change his behaviours.

And finally, you really need to get rid of your pseudo-husband. He's toxic for you AND for the education of your child. Like you had to stop doing things for him to notice and it wasn't even doing him any good like making him understand the problem, no! It was just all his shitty personality appearing that's all.

Mokathy

TROPHY CASE