Hello Chooseel

Phenol chloroform extraction followed by ethanol precipitation was the original manual method for purifying both DNA and RNA back in the 80s and early 90s when I first started in science

In essence for  both types of Nucleic acid, phenol removes lipid and protein and chloroform removes remaining protein and phenol: This just leaves essentially DNA/RNA and salt which you then clean up and concentrate by ethanol precipitation followed by a 70% ethanol wash. The latter desalts your nucleic acid essentially leaving a fairly pure nucleic acid prep

Now in the case of RNA purification you are meant to use ACIDIC phenol, NOT tris buffered (alkaline) phenol: With acid phenol during the initial extraction of RNA denatured (emulsified) protein as mentioned goes into the phenol and the upper aqueous layer contains primarily RNA and salt. The layer between the upper aqueous layer containing your RNA and the lower phenol layer containing extracted protein  - the so called interphase - contains lipid and most of your genomic DNA

Conversely with tris buffered phenol, when you perform a phenol extraction, protein is removed by the phenol but most of the RNA is sequestered in the interphase, leaving a lot of genomic DNA in the upper aqueous layer

In your case you could perform a phenol chloroform extraction and ethanol precipitation and then digest away genomic DNA with DNAse I but the concentration of your remaining RNA might be low

Regardless, yes manually prepared phenol chloroform extracted RNA is of sufficient quality if extracted properly to perform reverse transcription, making cDNA for RT PCR

Crucially in terms of quality and quantity of your manually purified RNA for RT qPCR you need to ensure

1. your 260/280 ratio is > 1.8: This demonstrates a high ratio of nucleic acid to protein and thus effective removal of protein by phenol

2. Your 260/230 ratio is > 1.0 and ideally 1.5: This indicates low phenol/chloroform and salt contamination of your final purified RNA; Such contaminants inhibit reverse transcriptase culminating in poor cDNA manufacture and thus poor PCR/qPCR results

If at all possible given that your phenol is the in correct pH for RNA extraction I would if at all possible re extract with acid phenol and chloroform followed by ethanol precipitation

Better still use something called Trizol to extract your RNA

However you choose to proceed the method for phenol chloroform purification of DNA and RNA; the latter for RT PCR is provided in the link below:

http://bitesizebio.com/384/the-basics-how-phenol-extraction-works/

 I have done this many times so contact with questions if you wish

Good luck !!

@Laurence Stuate Hall  thank you so much  but maybe, my question is not clear about the reagent. this reagent will be mixed with bacterial cells from culture broth (1:9V/V) after that centrifuge to harvest cells, discard supernatant, and flash freezing with dry ice+ethanol then preserved at - 80oC waiting for RNA extraction. there are other formulas of stop solution also but seem like this formula is easier to prepare so I wanna know how this formula work. anyway, million thanks. your answer is very informative.

Hi Chooseel

Im not quite sure exactly what you are proposing to do so cannot comment further. Do you have a copy of the protocol so I can examine and then comment

I shall say no more for now

Kind regards

Hi Laurence Stuate Hall

first line in topic of reagents and content in topic of cell harvest on first page

thank you for your kindness

http://www.pnas.org/content/suppl/2004/11/05/0407269101.DC1/7269SuppText.pdf

Hello Chooseel

I can find reference to phenol as a component of a stop solution but certainly based on the examination of the material you have give me i'm not exactly certain what reaction is being stopped or terminated; Perhaps I have missed this

Is it DNAse digestion by any chance ? Regardless, if it is an enzymatic digestion of some sort - forgive me for being vague - then a combination of phenol and ethanol would result in the following in effect terminating a reaction:

1. Phenol precipitates and therefore denatures protein. This includes any enzymes like DNase I that would be denatured and inactivated

2. Ethanol precipitates nucleic acid. If this for example applied to nucleic acid being digested then effectively the substrate would be removed from solution and therefore from any participating reaction

I hope that at least in principle this would give you some insight into how these chemicals might operate in the context of a stop reaction

If my comments in terms of what this stop solution is for, i.e. cessation of DNAse digestion apply then I can suggest alternatives. Please contact me again if you are interested but could you make it clear or refer me to the exact passages of where the 'stop' reaction applies

Hello Laurence Stuart Hall 

million thanks for explanation. Now I understand how it works. Like I said in question above, I spend time to find where original paper is but seem like no one cite the original paper for this reagent or just follow other paper. Anyway, RNase is more concerned in my case because of RNA extraction. I'm happy if DNase is contaminated lol However, Is it possible that 'stop' is to denature and stop RNase activity because of specific pH?

Thanks

Hello Choosel

Yes. maybe. But for certain if the stop concerns RNAase treatment then this is due to the phenol sequestering and denaruting the enzyme RNAse 

@Laurence Stuart Hall

Thank you  ^ U ^

Hi Steve

as far as I know is doesn't. That's why you have detergents like SDS in the initial lysis buffers. Having lysed cells with detergent acidic phenol then denatures cell proteins and also lipids. When you extract with acidic phenol proteins are partitioned to the lower organic phase; RNA and emulsified lipids, muco poysacharides etc are sequestered to the middle interphase and the top aqueous phase primarily contains RNA plus salts. Thus, you then extract with chloroform to remove phenol (which degrades nucleic acids with prolonged exposure and wash with 70% ethanol to remove salts: Leaving just RNA