First I would like to contradict the 4°C rule. With the phenolic compounds and chloroform RNases surely wont be active, so you do not have to be afraid of degradation. For phase separation and separation of RNA, protein etc into the different phases room temperature should be better.

I suggest to let it sit for 10 min at RT after Chloroform addition, vortexing and shaking vigorously, then centrifuge 15-20min at somewhere between 4000-6000 rpm (depending on the size of the rotor). Adding isoamyalkohol (IAA) to the chloroform in a ratio of 1:24 (IAA/chloroform) will surely help. We always have the mixture ready.

Hope it works like this.

Hi Teresa,

I have seen comparable phenomena in general with Phenol/Chloroform extractions of lysates to obtain nucleic acids. Often the reason is a high concentration of the lysate in all sorts of components, especially lipids and proteins (also depending on the detergent used for lyses - that does not apply to you, of course, becasue of your parallel samples). In any case, there are a few things you can try; let the extraction sit for 5 minutes prior to centrifugation and mix once a minute. You can also slightly increase the volumes in case your lysate is very concentrated in comparison to your parallel samples. Often, adding Isoamylalcohol (1:1) helps to get a sharper phaseseparation.

I hope this is of some help.

I don't know if it could help but in my mind you should centrifuge longer time as the phase appear slowly... I think it could resolve your problem.

Also are u running all ur centrifugation (initial ones) at 4 degrees. It is necessary that u run it in cold room.

Yes, I have experienced more or less similar issue when using TRIZOL LS for serum samples which seems to improve with the following conditions: Centrifugation speed (1300rpm), time (20min), and temperature set to (4C). Also, try to let samples stand for recommended times after adding kit lysis reagent and chloroform. I hope this of some help.

First I would like to contradict the 4°C rule. With the phenolic compounds and chloroform RNases surely wont be active, so you do not have to be afraid of degradation. For phase separation and separation of RNA, protein etc into the different phases room temperature should be better.

I suggest to let it sit for 10 min at RT after Chloroform addition, vortexing and shaking vigorously, then centrifuge 15-20min at somewhere between 4000-6000 rpm (depending on the size of the rotor). Adding isoamyalkohol (IAA) to the chloroform in a ratio of 1:24 (IAA/chloroform) will surely help. We always have the mixture ready.

Hope it works like this.

As per my knowledge, although 0.75 mL trizol is sufficient for 0.25 mL blood derivatives/serum, but sometime this criterion is not working due to depending upon the source of collection. You should decrease the volume of serum or increase the volume of trizol in your experiment.

Hi Teresa.

I often do lots of RNA isolations from different sources, but never from plasma samples. Nevertheless, most of the difficulties that I have found with some samples always end in the same problems regardless the source used. In the case of phase separation, from my experience I get better results if after chloroform addition I shake vigorously or vortex for ~15sec (control the time and don´t think that 15sec is just one or two shakes!! I think that for some samples this is actually a critical step.) then I incubate 10 min on ice and follow 15min centrifugation at 16'000g, 4ºC. These steps helps a lot in obtaining a clear aqueous phase containing RNA.

I hope this helps you. Good luck.

Hi Teresa,

I also vortex quite a lot the samples after addition of chlorophorm, often for up to 1 minute (I do it in a tube shaker though).

Regarding the need to centrifuge at 4°C, I am also not sure this parameter affects the clearing of the supernatant.

One thing that works like a charm and makes your supernatant retrieval extremely easy and precise is using the Heavy Phase Lock Gel tubes, see below:

http://www.5prime.com/media/3430/phase%20lock%20gel%20(plg)%20manual_5prime_1044378_032007.pdf

I hope this helps and good luck!

Forgot to mention that the HEAVY phase lock gel tubes work fine with regular Trizol and Qiazol, but you may need to pay attention if you use Trizol LS reagent.

In that case you may need a different type of phase lock tube.

!Hi Teresa!

We use the QIAzol Lysis Reagent and Chloroform for adipose tissue and always vortex and incubate at room temperature 5-10 min prior the centrifugation step. But for plasma RNA isolation, we use Trizol and glycogen.

I agree with most guys here. Let the sample sit for at least 10 minutes to form layers. Otherwise, most things I would have added are already posted here. Good luck!

use a phase lock as say Caterina

Thank you very much for all your answers, that really helps!

I already use the maximum µl that are possible, in my case 200 µl so at this step I can’t change anything. But I think you are right that I should vortex a little bit longer after the addition of chloroform. I used to vortex 15-30 seconds which is the maximum suggested in the protocol, but I will try it with 45 seconds. Thank you Anthony Beckhouse for the comment, I didn’t think about that, I hope 45 seconds are not too long. And I will also try a longer incubation before the centrifugation step (15000 rpm, 4°C, 15 min). I used to incubate for 2-3 minutes, but maybe 7 minutes on ice with vortexing every minute would be better (on ice is better because it is RNA?!). Thank you again for your comments, I will let you know how it works, but it can take some time, because I also have a lot of runs, where every sample shows perfect phase separation…

I am also agree with most of the guys the chloroform addition and incubation at RT for 10 minute and the vortexing should be gently not vigorously for 20-30 second shall be good as i followed,good luck

I have seen this problem, i do rna extraction too with trizol reagent, so you can add a few of reagents, and sure vortex very well your samples!!

Just to let you know: with the modifications I mentioned above, up to now, all phase separations were really good! Thanks again.

I'm sorry, but the problem is not solved.

Recently I did a RNA purification from a serum sample (I have more aliquots of this serum sample, all "treated" exactly the same way) and received only bad phase separation. The next day I used another aliquot and had perfect phase separation. Of course I used the same protocol...so I have no idea what's the reason.

Additionally, if it hasn't been mentioned yet, try centrifuging at 4°C.

Hello Teresa

I have exactly the same problem as you have and I am following the discussion as well. Till now, I tried several things (centrifuge for a longer time span (1hour) or at room temperature, increase volume of Quiazol and Chlorophorm (2-fold), vortex more extensively (45 sec)) However, nothing did work. I will let you know, if I find something to resolve the problem.

I got the same profile by using standard Trizol protocol. I could not find an explanation yet

Phase lock. For a dollar or two you save time and money.

hello,

what happened for your question? did you find an answer.

i recently have the same problem. any suggestion?  

Can anyone tell me which Chloroform from sigma is suitable for the miRNeasy mini kit?

It specifically says Chloroform (without added isoamyl alcohol) except the technical services suggested FLUKA chloroform but this contains isoamyl alcohol!

I had exactly the same problem and I also contacted Qiagen but they didn't help me either. After I tried several things without any good result, I decided to stop the experiments. A few months later, I resume the experiments and mysteriously the problem was solved (without any protocol modification). I don't know how to explain it, but I didn't have any problem until now.

I´ve tried using more chlorophorm (increasing 20% of the nitial volume) with miReasy serum/plasma Qiagen kit. Most of the time

We are currently using ZYMO DirectZol with very similar results bu without Phase separation.

I ran into the same problem today.

One out of 12 serum samples did not show any aqueous phase; I could only see two phases, the lower red phenol phase, and an upper phase that was totally cloudy.

I usually process 12 samples in parallel as we have the QIACUBE for automated RNA extraction, so I used the same conditions and reagents (miRNeasy Serum/plasma kit) for all samples. Following chloroform addition I vortexed all samples 15sec, let the samples sit at RT for 3min and centrifuged at 4°C for 15min at 12.000 g as recommendend by the QIAGEN protocol. 

I have processed around 60 samples (in sets of 12 per run as we use the QIACUBE for extraction) and never experienced this problem. Previously I usually shaked the samples instead of vortexing them. I wonder if this could be the reason.

Please let me know if you figure it out!

Hi Teresa - I was wondering if you ever resolved this? We are having the same issue. It worked fine, and now it isn't... It is always one or two samples that seem to remain cloudy and it is driving me crazy. We follow the Qiagen protocol (so add Qiazol lysis buffer, stand 5 mins, add miRNeasy spike-in, add chloroform, stand 2 - 3 minutes [tried more with problematic samples], centrifuge at 12000g for 15 mins at 4C). There seems to be no logic in when all is well, and when it isn't. It has been suggested i can try reextracting from the interphase which we might have to do for some samples but I don't know if that will work. I am just at such a loss and frustration - it is a waste of reagents and more significantly samples!

Thanks for any advise!

Sarah

I had this same problem recently, I see nice separation sometimes but other times there's almost no aqueous layer. I figured out that it has to do with the amount of buffer that your sample is dissolved in. If you add more aqueous buffer at the beginning then you may see that the issue resolves itself.

Hope it works :)

Today, running RNA isolation with TRI-Reagent BD I had this problem (cloudy phase without aqueous phase) and I finally solved it. I left my vials (sample+tri-regeant +chloroform) on ice for 15-20 min, followed by centrifugation at 12000g for 30 min. The results were so much better!! I hope you can solve your problem.

Hello Teresa

I think problem can be resolved by adding chilled chloroform and then it should be kept for 15-20 minutes on ice  and then centrifuging at 12000 g depending on protocolsin manual trizol method;second thing as you are working by considering Qiagen kit so be assured that your sample is appropriate in amount and lysis buffer is functional. 

I had this same problem again last week, but with only one sample (I was working with 20 samples)... I followed the recommendation from Lex G. Medina Magües but it didn't work. So, I returned the sample (yes, the whole solution with all phases) to the freezer (-80C) and after 4 days I picked the sample again to do another round of isolation and... guess what? No white interphase!! So, I think the solution is to put the samples in the freezer, but I'm not sure for how long. 4 days worked for me, but maybe less time it's enough.

I have solved this problem in three different ways, because sometimes one way does not work! I think that we have three posible solutions:

1) Add an extra volumen of Trizol (or Tri Reagen) AND  an extra volumen of chloroform. I used 950ul of Tri Reagen and 250ul of chroloform instead of the conventional 750:200 ratio.

2) Try freezing the samples with all phases and see what happens.

3) Pipette off the white phase (cloudy phase) without the organic phase, and split it into two new tubes and start the RNA isolation again; adding the conventional (750:200) Tri Reagent:Chloroform ratio. Then you can take 250-300ul of each aqueous phase and put it into one new single tube to complete the 500ul of the aqueous phase, followed by the isopropanol addition.

I used these solutions for RNA isolation from plasma and whole blood samples, because serum samples usually run correctly. I realize that whole blood samples are more difficult to resolve this problem.

Try these posibles solutions! And maybe you can combine these solutions to improve it or get a new solution to share!  Good luck!