I was using kits for RNA extraction but now I need to Isolate RNA by Trizol method. I have already tried some methods available online. But there might be some contamination because I'm getting nano-photometer reading around 1600 ng/ul.
Please suggest me any simple and effective protocol.
Dear Vishal, what exactly is your problem? The final concentration of total RNA varies and depends on the final volume of solvent you used to dissolve the pellet as well as the scale of your sample preparation. If your concern is about the contamination of genomic DNA, which possibly interferes with your downstream experiments such as RT-PCR, you can treat your preparation with DNase. If your nanodrop measurement tells an unusually low 260/280 value, which is frequently associated with the protein contamination, you can simply perform an additional round of phenol extraction to remove any residual proteins.
The use of Trizol for the isolation of total RNA is basically nothing more than a simple extraction procedure with acidic phenol followed by alcohol precipitation. Although its acidity forces most of DNA to be restricted in an interphase following extraction, I strongly recommend you to digest any residual DNA in your sample enzymatically, especially if your downstream applications involve PCR-based amplifications.
Dear all,
I'm using Plant leaf (Garlic) as a sample.
100 mg sample in 1ml Trizol
Homogenization
Addition of 0.2 ml Chloroform
Phase separation
Phenol:Chloroform:Iso-amyl-alcohol
Phase separation
Isopropanol Precipitation
Washing by 75% Alcohol.
Dissolve pellet in 50ul of RNAse free water.
Alternatively you can try Directzol Kit from Zymo. You have to load your sample in trizol into spin column, wash and elute.
http://www.zymoresearch.com/direct-zol
Trizol is the simplest and the most effective method to isolate RNA.
If you're getting small amounts of phenol carryover from the first aqueous phase removal, you can include an additional chloroform extraction (1:1). I find this often helps, especially with small sample volumes where removal of aqueous phase cleanly is...challenging.
Also, make sure your isopropanol precipitation is carried out at room temperature. Isopropanol precipitations are very prone to precipitating salt (especially guanidium salt) at lower temperatures so be careful.
I will ask you to add trizol, then follow by chloroform alone extraction then pelleting out your RNA, or if you want to increase the quality of your rna clubbing the trizol protocol with Kit method, column binding washing and elution will help you over come the quality problem, but quantity will be very less in comparison. So better opt, the trizol alone method, it works.
Since your sample is from plant, you have to use a good lysis buffer as well as an extraction buffer. or kit wise QIAGEN as a good kit available..
Hope you got some help!!
Use of DEPC treated water every where will help a lot in the quality step..
In order to get useful products it is crucial to get rid of any alcohol so be very careful in the final elution and drying. When you get the hang of it Trizol extraction is great but it will always be tedious...
Good luck
Contaminations are visible at e.g. 230nm (proteins/salts) 270nm(phenol) 280nm (protein). Check the ratios. A260 alone is not very helpful to determine RNA quality. Many issues during TRIzol extraction can be solved by careful and conservative withdrawal of the aqueous phase.
the Trizol method can be conveniently combined with direct spin column clean-up of the lysate, see http://www.amp-tec.com/assets/downloads/RNAready%20Add-On.pdf
it even includes DNase for on-column digestion, which can be important for sheared genomic DNA in your sample.
I have used Trizol method and for me was easy and faster than older techniques like isothiocyanate . If you dont want to use this method you can buy a kit of molecular probes, they are good. It is also important to take into account all the procedures before to get RNA, like the use of gloves , the sample extraction in good conditions without contaminants, you can also work with ice to avoid RNA degradation. Make the procedure as soon as possible and you can leave the procedure in the precipitation with isopropanol for the next day, store the sample at -20ºC, and then continue for the last step. I suggest you to elute in a mínimum volume like 20 ul. but it depends of your sample, otherwise you will get low concentration. It is easier dilute than concentrate. The ratio A260/A280= 2 . After you have finished you can run a agarose gel with MOPS in DEPC to probe the integrity of the RNA. Good look
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA