A monophasic solution of guanidinium isothiocyanate and phenol is known as Trizol or Tri-reagent provides stability and integrity to RNA during isolation. Commercially, from company to company the name varies but the composition remains the same.
The highly unstable nature of RNA and the predominant universal presence of RNase enzymes are the two most common factors that make the RNA isolation process sensitive and painful. Anyway, the principle, procedure, protocol and importance of RNA extraction are explained in our previous article.
Let me explain in short, a high yield, pure and stable RNA is a key requirement in gene expression, pathogen detection and viral identification process. Thus, the entire transcriptomics studies rely on the RNA, and how it is extracted.
I have years of experience in nucleic acid isolation. In my opinion, RNA extraction is a bit more complicated, complex and hard process than DNA isolation. Notwithstanding, the chemical composition of both types of nucleic acid isolation is relatively similar.
Immense experience and high-end expertise are required to isolate RNA. Tons of optimizations and protocols are there on the internet but the problem is, that no protocol is reproducible, every time, the lab personnel has to adjust things accordingly. Automated RNA extractors are excellent options to achieve every goal of RNA isolation but are relatively costly. So laboratories have to rely on the expertise of the scientist.
The use of traditional processes and conventional chemicals are, however, effective, cheap and reliable options for any nucleic acid extraction process.
I am talking about the phenol-chloroform. The isolation process becomes even more effective by adding guanidinium isothiocyanate. Trizol is the registered trademark of Invitrogen and is used for RNA extraction.
But what is the exact chemical composition of Trizol or Tri-reagent and how does it work in RNA isolation? Let us find out.
The main goal of any RNA isolation procedure is to achieve excellent RNA quality & quantity and preserve RNA integrity.
Key Topics:
What is Trizol™ or Tri-reagent?
Trizol is an RNA extraction reagent that comes up with phenol, guanidinium isothiocyanate and other essential chemicals, although the company hides the name of other chemicals.
Other companies have the same formula and composition with other brand names like Tri- reagent, QIAzo, TriSure, RNAzol and TriPure. It is a monophasic solution that stabilizes and improves the yield and quality of RNA.
Monophasic:
Liquid solvent prepared with two or more components in one phase.
Qualities of Trizol:
- Effective RNase inhibitor
- Helps in tissue homogenization
- Cell membrane and nuclear membrane disruption
- Deproteinization and protein digestion
- Nucleic acid separation
- Prevent RNA degradation during purification
Ideally, it is a suitable and commercial modification of the one-step protocol of Chomcynski and Sacchi (1987) and used to isolate RNA from humans, plants, yeast, bacteria, viral or any other tissue.
Note that it can not only isolate RNA but DNA and protein too. Thus used in those procedures too, but it gives promising results in RNA extraction.
Principle of Trizol (How it works?):
Trizol with phenol and guanidinium isothiocyanate effectively separates DNA, RNA and protein. Phenol separates nucleic acid while the guanidinium isothiocyanate works as a chaotropic salt and denatures proteins. In addition, the acidic nature of the reagents separates DNA from RNA in two different zones.
Homogenization by Trizol followed by chloroform treatment separates RNA, DNA and protein in the upper organic phase, interphase and lower phenolic phase, respectively. RNA from the upper aqueous phase is collected and precipitated by any alcohol.
Advantages:
The present isolation technique is
- Comparatively cheap and high yield
- Can isolate RNA from any tissue
- Removes RNase enzymes
- Stabilizes RNA
- Protect RNA from degradation
- Store RNA and prevent its integrity
Limitations:
RNA isolation using the Trizol reagent is a
- Tedious process
- Need centrifugation and thus increase isolation time
- Need technical skills and expertise
- Can’t work for all tissue types and thus results in poor lysates, sometimes and for some hard tissues.
- It can’t work effectively for many samples.
- Can’t give uniform results
- Phenol is volatile and can harm the personnel.
Protocol of RNA extraction by Trizol:
- Perform all the pre-preparation before initiating the experiment.
- Take 50 to 100 mg tissue, add 1ml Trizol reagent and homogenize tissues well.
- Incubate the homogenous tissue mixture at room temperature for 5 to 7 minutes to dissociate the nucleoprotein complex.
- To the mixture add 0.2ml chloroform per 1ml of Trizol reagent, vertex the sample properly and mix it by inverting it several times. The sample can be mixed by vigorous skating for 20 seconds.
- Incubate the sample at room temperature for 5 minutes.
Note: Do not open the sample cap outside the laminar hood.
- Centrifuge the sample roughly at ~5,000 rpm for 15 minutes, and remove the tube without disturbing it.
Note: Perform centrifugate at 4°C.
- Carefully collect the upper aqueous phase without disturbing the interphase in a sterile tube.
- Add 0.5 to 1 ml isopropanol (75%) (chilled) and incubate the tubes at -20°C for 1 hr or overnight.
- Inverts the tube many times until the RNA precipitate appears.
- Centrifuge the sample at 10,000 rpm for 2 minutes to settle the RNA pellets.
- Collect the pellets and wash 2 to 3 times with chilled alcohol (ethanol).
- Resuspend the pellets in 50 to 80 µL of RNase-free water and incubate at 60°C in a water bath for 10 minutes.
- Once the pellets dissolve, run the sample on agarose gel electrophoresis to investigate the quality of RNA.
- Also, perform RNA integrity assay and quantification before using the sample in downstream applications.
Note
Like other RNA protocols, you can also use the DNase and proteinase K to remove DNA and protein contaminants in the sample, if required.
Common suggestions:
- Use a standard RNA extraction setup and handle the sample accordingly.
- Prevent RNase contamination by taking necessary precautions.
- Trizol contains phenol which is toxic so it is highly advisable to handle it with care, and take training before handling it.
- Perform all the steps under an aseptic hood and at 4°C, when special conditions are not mentioned.
This article may help you to learn more: 21 Things to know for Effective RNA Extraction.
Wrapping up:
RNA isolation, as I always say is a tedious and painful process. The results are always uncertain but when you gain substantial experience, you can isolate RNA effectively, however, do follow safety measures and RNA extraction standard guidelines.
Especially when you are isolating RNA from contagious RNA viruses like the coronavirus, take extreme care and follow standard SOP.
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