I haven´t used the protocol but according to your reference they add the bleach before heating in the microwave (" into a TAE and agarose mixture prior to melting the agarose"). I am responsible for the safety-at-work in our lab and I wouldn´t adopt this method because bleach will of course evaporate during the heating and harm your lung if you inhale the air in front of the microwave.

In our lab, we do: agarose gel and you do your electrophoresis. At the end of your migration, you put your gel in the bath of "RedSafe" reagent (we use gelRed -10min) than water bath (10min), then uv lamp. All those reagents has to be RNAse free...

Another quick method is to heat 0.5ug RNA to 65-80degC for 10 minutes before loading it into a standard 1-2% agarose gel.   I usually run the gel after the DNaseI heat-inactivation step (samples are cooled down on ice or immediately frozen first).

Yes Have tried with Bleach,we add it before microwaving.Got good rna bands.

We use Etbr 

Update on the bleach gel!

I added the bleach just before pouring the gel into the mould so I assume there was no evaporation of the bleach and thus full denaturation potential (don't know if it's relevant...). I did not add RedSafe to the gel, I chose to dye the gel after electrophoresis. However I couldn't incorporate RedSafe efficiently to the DNA on the gel: I had to add a lot of it (3x the recommended quantity), leave it for over an hour and I couldn't rinse the gel afterward as I completely lost the staining! I still managed to make a decent picture of my gel. The bands were sharp and very neat, however, the staining was not adequate... I blame the RedSafe!... I might try again, adding some RedSafe directly to the samples... and stain afterward too!

Would "bleach gels" work in order to evaluate the size of an RNA fragment (run next to RNA ladder)?

Tx

-J-

PS:

I know this sounds like a stupid quetion but I read that "Bleach" gels can ber used to assess the quality of RNA (degraded or not) but I find nothing saying that the RNA will be migrating properly in order to asses its size when comparing with RNA ladder (some secondary structures may remain and thus prevent this).

Thanks again...

@Julien Dal Col:

The original paper said they "postulate that the 6% sodium hypochlorite present in commercial bleach denatures the secondary structure of RNA by destroying hydrogen bonds..." However, the paper did not provide any figures or information addressing RNA denaturation in the bleach gel. So I too am curious to find out. 

@Marie France Bouchard: 

Have you had any further success? I have tried running a bleach gel by adding bleach before microwaving since the paper said that the bleach did not mix properly with the agarose after microwaving. Did you have any issues in mixing the melted agarose with bleach? 

This protocol did not work for me. I have tried several times, and I created a new protocol (simple) to analyze my samples. You just add 2 volumes of 1M Guanidinium thiocyanate to  the sample, and heat up 5 minutes at 65ºC. You can load and run in a conventional 1% agarose gel.

Works fine with the protocol just like described in the paper, and with GelRed 5000x. DNA Ladder shows a bad smiling, but that was to be expected as GelRed should be used with the post run staining protocol (too much work for me though). Ribosomal 28S and 18S bands migrates around ~ 1.4 and 0.8kbp. I might try EtBr in the future, and save further

I tried it, exactly as instructed in the methods of that paper and it worked really well, see attached RNA gel. TBE buffer, Regular 6x loading dye, Ethidium Bromide, 1% bleach. Electrophoresis: 4hrs, 4C and at 50V. MB

Based on your ladder, though, it looks like it isn't running by size?

Chlorine (from Bleach) can react with the platinum wire in the Electrophoresis tank. I ran a "Bleach agarose gel" with slight modification from Article Bleach Gel: A Simple Agarose Gel for Analyzing RNA Quality

Although I could see two RNA bands on my gel after staining with EtBr, there were many downside of using this methodology. First of all heating (boiling) bleach with agarose could be hazardous, so I mixed bleach only after the agarose was melted in 1X TAE buffer.

Firstly, I mixed 6 ml of (6% stock) bleach solution to 60 ml of melted agarose gel and it produced a lot of air bubbles in the gel. For the second time, I added only 2 ml of bleach (6% stock solution) immediately after meting of agarose gel and it was bubble free.

I ran the gel at 70 V for about 3 hours and I was surprised to see brick colored brown precipitate at the anode end (+ve electrode) of the electrophoresis tank. I suppose this was due to a reaction between Platinum wire and bleach. I used 1% agarose gel. I found a research article that talk about oxidation of platinum by chlorine. Article Oxidation of Chloride Ion on Platinum Electrode: Dynamics of...

I don't think this procedure works better than formaldehyde gel electrophoresis since this is hazardous and can damage your electrophoresis apparatus.

It does not work better, it is easier and cheaper. I have never had problems heating agarose with bleach, so not sure how that could be azardous. You have to melt the agarose not overboiling. It is not supposed to realese fumes in the room... you are not suppose to feel it at all. I do not think it is damaging the apparatus at the concentrations of the protocol, certainly it is not supposed to give any brown precipitate. Please try the method sticking to the protocol, it is clearly specified not to add bleach after melting the agarose. Works very well for a lot of people including me

Guys. Stop using EtBr. It's 2018. Get with it. Use less toxic staining reagents.

Casey honestly you are a bit off topic here. You can start another topic where you explain us why in 2018 we should give up EtBr, while we are using commonly formaldehyde, xylol, polyacrylamide, DEPC, azoxymethane, trypan blu and so on. Actually, there is already a topic https://www.researchgate.net/post/EtBr_is_a_carcinogen-is_it_a_myth2 , so no need to hijack this one. Anyway, EtBr in 2018 works very well, it is dirty cheap, and probably less toxic then dozen of other things used to much higher concentrations and amount. Obviously using gloves and being careful is important , but paranoia doesnt help. If you are lucky to afford some expensive alternative good for you, but give the others a break.

To contribute with an answer similar to that of Paolo Tenti and others. I use the protocol as stated in the paper, but add the bleach at the same moment when I add the EtBr i.e. when melted agarose is tempered (regular safety measure for not liberating fumes with EtBr). For added precautions I cast the gel in a fume hood, and replace the TAE 1x from the electrophoresis chamber every 2-3 of my gels (and rinse the chamber with distilled water, common procedure). The same equipment is used for other routinary DNA gels, and TAE buffer was prepared with regular distilled water, thus it should be the paradise of RNases. The method is really cheap and quite safe, it helps to save time as you don't need some additional set of electrophoresis chamber, casting tray, combs, agarose, or to prepare buffers with ultrapure RNase free water or DEPC water.

Thanks Juan for really nice contribution. I have always been super careful with RNAses, using DEPC etc but the paper clearly shows the protective effect of the bleach in presence of RNAses, and you seem to confirm it. Great to know we can add bleach at the same time than EtBr, I have to try it and maybe I will also try to use not treated water. DEPC is very much indeed a nasty chemical, possibly to replace with inexpensive dimetil dicharbonate. Glad to know untreated water is just fine

Paolo. While I appreciate your mansplaining and condescension paired with entertaining grammatical errors, I do not need your opinion on my addition to this thread.

I came to this thread to understand the compatibility of these gels with SYBR safe. However, most of you folks seem to be stuck in 1980, so I did not find what I am looking for. So yes, addressing your outdated staining practices was entirely relevant to my search.

Being a white male and therefore prone to unbased self-assuredness, I can see how you might have missed this, but EtBr is entirely too toxic for women to use. “Dirt” cheap or no. Period. Women have much less of a voice in biotech, and so its use is perpetuated in some antiquated labs. There Is a reason, however, that most reputable research organizations outlaw it outright.

if I need to convince you of the dangerous effects of EtBr, perhaps you need to focus less on the relativity of my answer and more on the fact that you are 20+ years behind the rest of the science community when it comes to simple agarose gel staining. There is no need to further discuss EtBr’s toxicity as it is well established.

Let’s try to use some gender neutral reagents so that our answers apply to all of Research Gate’s users, shall we?

Anyone else ? Results with better pre-cast stains a la SYBR safe ? Again, not interested in results using EtBr

Also, RNAse Zap works wonders, is an irritant upon skin contact but overall very safe, and can be made in-house to save money (though it’s not terribly expensive commercially). I extracted rna from dozens of pigs on 3 occasions in a homemade lab in the literal jungle of Guatemala. I didn’t RNAse contaminate a single sample. Use liberally - to the point where you see a sudsy residue on your pipets, work surfaces and gloves. From there, using the bleach gel discussed in this forum (i.e to QC) will protect your samples from contaminating RNAses in your gel box/cast/agarose/buffer quite effectively.

Cheers

I think this blog entry about EtBr may be relevant, as the writer is an organic chemist.

From the text: "The reference above found that the SYBRsafe alternative was actually much more toxic than EthBr to the bacterial cells used in the mutagenicity tests. SYBRsafe was toxic at concentrations as low as 1 microgram/ml, whereas EthBr toxicity was not observed until 250micrograms/ml. The authors suggest that this is because living cells are much more permeable to SYBR green than to EthBr."

https://blogs.sciencemag.org/pipeline/archives/2016/04/18/the-myth-of-ethidium-bromide

I have used EtBr, SybrSafe, and RedSafe. I don't like SybrSafe at all, it does not provide clear neat bands. I prefer RedSafe/GelRed, seems to be less "toxic" than EtBr, and works equallly with UV light. I will post here when I use a bleach gel with RedSafe staining.

Here is some actual literature as opposed to some guy’s blog: Article Comparison of SYBR (R) Green I nucleic acid gel stain mutage...

.... published in 1999. Keep up ;)

Here we go, Casey hijacking the thread again. I ll just ignore your petty personal attack on the grammatical mistakes; whatever you say, all your rant and your reference are OT here. Be mature enough to clean all your mess in this thread, go to the EtBr toxicity discussion if you like, then come back as a respectful human being to discuss about possible variations to the protocol which is object of the topic. Thanks

I've used the protocol with SYBERsafe, and it works. However, as Juan stated, the bands aren't great. My department has banned EtBr and I don't have the time or energy to fight it, so I would love to hear more about RedSafe staining.

Anyone who had good bands on a bleach gel using SYBRsafe? The protocol looks pretty cheap and easy however, just wanted to make sure whether it gives a similar result with SYBRsafe.

Yes, when I have good RNA I get good bands. The RNA I had referred to above was slightly degraded.

Thank you Douglas. That clears up the doubt I had.

I also tried with SYBERsafe. My intensity is also too low.

Just tried yesterday, the RNA bands were much weaker than the DNA ladder I used on the gel. I used Apex safe, which I assume is similar to SYBERsafe. The gel looks messy. Will have to explore more on quick RNA gel protocols.

I just got the RNA bleach agarose gel work and thought it might be helpful to someone following this post.

I used the easy recipe (1% v/v commercial bleach in the gel) from this article with a little modification.

Article Bleach Gel: A Simple Agarose Gel for Analyzing RNA Quality

When I used a DNA loading dye as suggested by the authors, my RNA degraded. Then I ordered RNA ladder and RNA loading dye and followed the manufacture's protocol in preparing RNA-dye mixture. This protocol is working beautifully. Attached is an image of my RNA gel prepared with TAE and stained with Apexsafe (similar to Sybrsafe). The first 3 lanes next to the ladder have RIN of 10, 9 and 7.4 sequentially. I've repeated several times and it does the trick every time.

Hi there, just to share an alternative method for RNA agarose gel using H2O2. Haven't tried it yet, but looks like this method is better than the bleach one, as they compare in the paper: "Hydrogen peroxide agarose gels for electrophoretic analysis of RNA" (2017)