hi Abhishek Dubey

What your mentor said is a precautionary step while performing Trizol based RNA extraction. There is NO MYTH or ANYTHING WRONG in it.

1. Yes, do not mix harshly after the addition of Trizol to the cells.

2. Yes, do not vortex after adding chloroform, just flip your Eppendorf tube up and down 7-8 times.

3. Yes, do not pipette vigorously, use 2-20ul pipette just to mix. Anyway, you will add just 10-20ul DEPC water.

Regards

Saurabh

If the points are precautionary of not, but sure they are not a myth. The points made are most probably a part of the protocol used in respective lab that is followed by all the members. This minimizes the chances of explainable differences in the results from different experiments.

Further,

1. When resuspending cells in Trizol, they can make clumps. Do not pipette vigorously and cause bubbles in the solution. Also, do not use syringes. Is this precaution really true ?

>> Not always true and depends on the cell type and how the sample is being processed. For some sample types resuspension with vortexing is preferred and for some not recommended. Bubbles can cause unnecessary spill or excess volume which can lead to contamination. Syringes can be chocked with cell debries.

2. Do not vortex after adding Chloroform as that can lead to genomic DNA contamination. Is this precaution really true ?

>> Phase separation is a very important step and one must be careful to not disturb phases and this could greatly influence the quality and quantity of the final eluent.

3. Do not pipette the water/buffer when resuspending RNA pellet. This can shear the RNA ! Is this precaution really true ?

>> Sure, vigrous pipetting/vortexing can cause sheer or even formation of inclusion bodies.

The precautions in any protocol are meant to minimize the errors and for uniformity. Also, these precautions are based on experiences and primarily on trouble-shooting. If you have doubts, you can simply try not to follow these precautions and can evaluate the quality and quantity of your final product.

Oh, I guess I'm going to be the awkward person who disagrees with the previous posters. Sorry, previous posters.

1. Cells should lyse completely in trizol, that is...literally the point of trizol. It contains phenol and guanidium isothiocyanate: it's basically a maximally unfolding/disrupting/denaturing mixture.

You shouldn't need to pipette up and down, because most of your cell pellet should be near-instantly dissolved. If you need to vortex, vortex. It's fine. Everything is getting burst/disrupted/unfolded/solubilised, and that's fine.

For reference, I frequently use powdered muscle tissue, and I blitz the hell out of that after adding trizol. Even after all this, some material doesn't solubilise, because muscle tissue is so protein rich (and most of that protein is fibrillar) that not all of it can unfold fully. I spin this insoluble material down before proceeding, but if you're starting with cell pellets you should not need to: there should be no insoluble material.

2. No, vortex the hell out of it. Genomic DNA ends up at the interphase, so if you want to minimise gDNA contamination, just be very cautious removing the aqueous phase. Or design primers that span large introns (for qPCR) or DNAse treat after precipitation (for other applications). Good phase separation benefits from good homogenisation. I usually vortex for 15 seconds.

3. RNA should dissolve incredibly eagerly, so this should not be an issue. Also, shear stress usually applies to gDNA-grade lengths of nucleic acid, rather than mRNA-grade lengths. Chromosomes can be single molecules several centimetres in length, and that's a very long string to expect to survive vigorous vortexting. In contrast, most mRNAs are at best a few microns in length, and those will usually survive vortexing just fine.

For reference, I typically add my DEPC H2O directly to the pellet, wait a minute or two, then vortex briefly (~5 sec) and collect everything in the bottom of the tube by a quick (pulse) centrifugation.

John Hildyard your explanation do not differ much except the fact that you used a more aggressive wording and did not consider the fact that you being an experienced researcher telling a new student to not follow the protocols. Further, David Farringdon Spencer

Is it just to recommend vigrous vortexing without knowing what is the cell type of what is the extraction protocol? Total RNA or mRNA?

Because according to the previous argument, RNA must be immune to vigrous vortexing and does not matter which is the state, one can blindly vortex? Why do sequencing facilities1 recommend not to vortex? Why do commercial kits use words like carefully/gently in their protocol or mix by inversion2,3?

Who in the world want to have degraded RNA but still there are often problem of RNA quality? You can also argue on why to place RNA on ice, as RNA is stable enough to not degrade at 20-25 degree Celsius. This must also be an ignorance?

Further, for a critical experiment, if careless handing and protocol without precautions ruined the sample, who will come to rescue and whom to blame?

Please add in following posts (which you will surely post to defend your previous posts) your experience about always means 100% success rate is nucleic acid isolation specially when you have not taken any precautions or followed any protocol.

I respect and support your experience and explanation, but using words like non-sense, ignorance etc in this particular case is discouraged. One must take into account the academic level of the person. For example, Abhishek Dubey is very diligent and therefore questioned the instructions. But his friend XYZ , at masters level, is not so careful. In this case, the instructor gave the same protocol with precautions) to both because they both work in same lab. Is the instructor moron/non-sense/ignorant that he gave the protocol with precautions? Isn't this a learning process where Abhishek Dubey found doubts which lead him to learn more from experience people like you John Hildyard & David Farringdon Spencer

Abhijeet Singh : all good questions, and I did try to be as polite as I could.

Let's take your RNA_Trizol example first (3).

First, this is a protocol for cancer samples (which are often tough), accordingly, it recommends Homogenization with TissueLyser, in other words, chuck a ball bearing or two in with your sample+trizol and vibrate it on a giant shaker until everything is blended.

That's...pretty strenuous, harsh treatment.

For phase separation? "Cap sample tubes securely and shake vigorously by hand for 15 seconds" -that's basically vortexing with added cardio & upper arm workout. So yes, vortexing is fine for phase separation steps.

The only steps that say "do not vortex" are the precipitation step, and the wash steps and those...were not something asked in the original question.

Personally, I'd agree with both of those: you don't _need_ to vortex during precipitation, since you're mixing isopropanol and water, things that inherently mix really well. Pipette or inversion is fine.

For washes, you don't vortex because vortexing knocks the pellet off the side of the tube and possibly breaks it into tiny flecks of RNA which are a massive pain to avoid accidently discarding. Gentle washes can remove salts without dislodging the pellet, and that makes everything easier.

They don't recommend vortexing for dissolving the pellet, but honestly, it doesn't hurt (as noted, I do it routinely). What they suggest (flicking) should work fine, but they also suggests incubating the RNA in water for up to an hour at 60, which...yeah, that should almost never be necessary.

As I noted, in most cases all you need is "add water, wait a minute or two, done".

They also recommend not vortexing during DNAse treatments etc, but that's because these are protein-based reactions, and vortexing enzyme mixes usually just gets a ton of foamy protein bubbles and a crap, inefficient enzyme mix.

The RNAseq protocols seem similarly happy for peeps to run stuff through tissuelysers and then shake like hell, but are oddly opposed to vortexing. I leave it to you (and readers) to decide whether shaking something using your hands (or a machine) is fundamentally different from shaking something using a machine (but I can state that in my experience, both are fine).

Your other link (the agilent one) is for phenol/chloroform/isoamylalcohol RNA isolation, which...again, isn't what the original question concerned, at all. PCI is a really old-school method, and is a bit of a pain in the backside, which is why folks tend to use trizol.

Also, at no point does it say "do not vortex", and in the troubleshooting for RNA resuspension, it suggests "vortex the pellet".

So, yeah.

I am trying to be helpful, here. I've been doing this for...quite a while, and RNA work is definitely a field with a lot of superstitions and myths, many of which are founded on simple misunderstandings.