When using TRIzol (or the similarly-named products), getting the aqueous phase without organic phase contamination is more important than getting all the aqueous phase.  That said, there are some things I can recommend to improve your purity:

1. I actually perform the chloroform step twice to separate any organic phase I accidentally picked up from the first time.  This can lower your total RNA yield, but pure RNA is better.

2. I leave my samples in 2-propanol at -20 degrees C overnight. This has improved my yields.

3. Zymo Research has a Direct-Zol kit that you put the TRIzol+sample into, and, in my experience, they work very well and give pure, concentrated RNA samples.

In response to Petra, I used TRIzol with a kit because the fatty tissue I was working with was not broken down as efficiently by the kit's buffers, but TRIzol extraction alone did not yield pure enough RNA samples; the combination worked best. Perhaps it's not best for everyone, but TRIzol has its uses.

What does your A230/260 ratio look like? When you isolate your RNA, do you also do a DNase step?

When my RNA samples have shown to have contamination, I have done phenol/chloroform extractions to "clean-up" the samples and make them more concentrated. Then I usually re-spec my samples to ensure that the A260/280 AND 230/260 ratios both look better, before starting with the RT process.

How do you isolate RNA? have you use RNAeasy kit or trizol or home-made reagents?

How sharp is your pick? In some case low A260/A280 ratio can come if RNA did nit fully dissolve. Or you have not correct pH of the buffer. If you are sure that you have protein contamination, you can use even chlorophorm cleaning, and thereafter, precipitation with ethanol. 

Hello Taras Sir, I use trizol reagent to lyse the cells and subsequently proceed for RNA isolation using chloroform and 2-propanol precipitation method.

Can you suggest me steps for chloroform cleaning of RNA samples?

Dear Mansi,

Please have a look at the attached files

It is much more likely that you have Phenol contamination from your Trizol extraction than protein contamination. This is quite common unless you are really experienced with doing the phase separation. Phenol contaminatioon IS a problem with downstream RT, but you can rescue your samples by doing additional chloroform extraction followed by ethanol precipitation and several washes with 70% ethanol before drying&resuspending.

I do hope that you are aware of the fact that Trizol is 38% Phenol, of how toxic Phenol & Chloroform are, what to do if you do get Phenol on your gloves (it can permeate both latex and nitrile ones!) and that they should be used in the fume hood and that you are not allowed to use them if you are pregnant (and not a good idea if you are trying to get pregnant). For example

http://users.ox.ac.uk/~phar0036/biomedsafety/labsafety/chemicalsafety/phenol.html

I personally don't understand why one would use Trizol when you can reliably get excellent RNA quality from kits like the RNeasy one in a lot less time. And I've done my fair share of Phenol extractions (of radioactively labelled aptamers).

As Anne was saying, it is absolutely essential that you have some way of getting rid of genomic DNA contamination (either by DNase digestions or using specific DNA away buffers when setting up your RT). If you still have genomic DNA in your downstream PCR (I assume you do RT-qPCR) then you can get amplification from that even with intron border spanning primers.

When using TRIzol (or the similarly-named products), getting the aqueous phase without organic phase contamination is more important than getting all the aqueous phase.  That said, there are some things I can recommend to improve your purity:

1. I actually perform the chloroform step twice to separate any organic phase I accidentally picked up from the first time.  This can lower your total RNA yield, but pure RNA is better.

2. I leave my samples in 2-propanol at -20 degrees C overnight. This has improved my yields.

3. Zymo Research has a Direct-Zol kit that you put the TRIzol+sample into, and, in my experience, they work very well and give pure, concentrated RNA samples.

In response to Petra, I used TRIzol with a kit because the fatty tissue I was working with was not broken down as efficiently by the kit's buffers, but TRIzol extraction alone did not yield pure enough RNA samples; the combination worked best. Perhaps it's not best for everyone, but TRIzol has its uses.

John,

Completely agree that Phenol extraction has its uses, but IMHO should not be the default method for RNA extractions.

Most of the time the argument for using it is "this is how we've always done it" or "it is cheaper than the kits". It really isn't cheaper though once you calculated time investment particularly when processing more than 4 samples, the cost of chemical waste disposal which is not covered by University overheads here in the UK, so every lab has to pay for it themselves; the fact that inexperienced people often cannot get good quality RNA with it and have to repeat experiments to get another set of samples...

IMHO "this is how we've always done it" is not only problematic because if there are better or more reliable methods around, any self-respecting scientist should be willing to learn and use them; but also because avoiding the use of dangerous chemicals wherever possible should be encouraged. Risk assessments for the use of dangerous chemicals are required here in the UK under Health&Safety regulations.

In practice, I've seen too many students not even aware that they were handling dangerous stuff such as Phenol or Chloroform and exposing themselves and other people in the lab to it unwittingly (for example other female PREGNANT staff).

I completely agree with Petra about phenol, it is toxic compound and RNA-easy  kits can give you faster and easy results. BUT one should consider that conditions in India and in UK are totally different. In some University it is not so easy to order such kits. Similar is for the phenol waste: the regulations rules about this may be very different in different countries. 

Of course, DNAse treatments is a primary importance for RT-PCR and one have to use also as a control primers what can amplified only form DNA, but not form cDNA to be sure that DNA contamination is absent.

Phenol/chloroform does the trick, yes it's nasty so do it in the hood and be extra careful. I would add 1 volume phenol/chloroform to your sample, vortex and then spin to separate the phases. Precipitate the aqueous phase with 2 volumes of 100% ethanol at -80°C for at least an hour (this may be superstition but always worked best for me), spin hard, wash with 70% ethanol, air dry and re-spec. 

Hi Mansi,

I am using Trizol for extraction of RNA from cell culture and I have done a lot on human WBCs. As John T Corthell mentioned above, 

Hi Mansi,

lets split your problem into 2 different situations.

1. if you have important RNA sample (and are not able to re-isolate RNA from the initial material) and need to purify it.

you can have different contaminations of your sample - genomic DNA and/or protein(s), and If you are not sure what exactly you have in the RNA (in most cases) - you will have to remove all of them (DNAse treatment and/or proteinase(s) treatment, appropriately).

However, there are other contaminants (interfearing with the subsequent RT-PCR) almost in all RNA preparates - polysaccharide molecules (mainly proteoglycans, sometime heparin or hyaluronic acid). It is mentioned in the TRIzol instruction by very small letters as additional information.

And it is the most complicated situation - these molecules originally go in the same water phase as RNA. Even if you will remove gDNA and proteins, you still will have them (high 230/260 ration is an indirect sign of their presence). Unfortunately, there are no simple and reliable/cheap methods to degradate these molecules.

So, in such situation I think the best way is not to remove all possible contaminants but bind your RNA onto DNAse treatment column ( please, mention - not DNAse treatment in solution!) getting away inpurities. This approach will combine few effects - gDNA degradation (by DNAse), remove of most protein(s) (they almost don't have affinity to the columns) and some proteoglycans (at least, a part of the molecules will go through the column).

You can find different kits for this puprose, all work well. I personally prefer from Ambion - this company is concentrated on the RNA methods/reagents only and their products are the most reliable in the filed (also, you will find many useful tips at their web-site)

http://www.lifetechnologies.com/se/en/home/brands/ambion.html/

2. if you can re-isolate total RNA from the initial sample

there are some principally important points

-VERY important to get a right amount of the starting material (ratio sample/TRIzol volume)!!! See TRIzol instruction, and divide the starting amount by 2 - you will get less RNA but it will be much more pure (even without DNAse treatment it will work for RT-PCR). If you need a lot of RNA - increase amount of TRIzol accordingly.

-separate phases (by centrifugation) very well (at high-speed)

-take only water phase (as colleagues adviced) and leave some water phase over phenol layer.

- precipitate RNA-centrifuge-wash with 70% ethanol-remove all liquids thoroughly (remove by pipette - not pouring! and slowly)

-not overdry your RNA pellet and dissolve thoroughly (pure RNA will be transparent - not white!), keep on ice short time and -70-80C longer (not -20C)

Hope, this information will be useful for you and good luck with your experiments!

It will also be prudent enough to check the pH of trizol especially if it is not the commercially prepared one. Acidic pH of the trizol plays a role in denaturing your protein present at the initial stages of RNA isolation from tissues.

However, if you still have contamination after dissolving of RNA pellet in nuclease free water, then sodium acetate precipitation of the RNA followed with redissolving in DEPC water is a great step you should consider.

Hope this helps.

I am currently having the same problem (used the Trizol method) I dont have any sample left to redo the RNA purification. Can I run my 50ul samples over the RNA mini kit again?

Brittni,

quite a few people do that to get "extra" clean RNA - you'll loose some RNA, but should end up with enough to do it. I would always attempt to only use < half of the tissue for each extraction (if you have enough tissue available, but should be if animal tissue). Just imagine what happens if you dropped your RNA column/prep during the clean-up or PCR set-up...

Not an answer, but a follow-up question - I hope, that is ok?

I extracted RNA from c. elegans recently using the classical Trizol method (there is some risk that the columns in the kits will filter away the type of RNA we are looking for). The purity is fine for young worms, but worse for old worms.

Is it possible to put the sample through the Trizol extraction again?

And if, will I start from the phenole chloroform step to separate the phases? (I read some just repeat the precipitation with 2-propanol, but I don't quite understand why this will help me get rid of protein contamination?)

Thanks in advance for your input!