4-1.4.1. Organic extraction method
This method involves phase separation by addition and centrifugation of a mixture of a solution containing phenol, chloroform and a chaotropic agent (guanidinium thiocyanate) and aqueous sample. Guanidium thiocyanate results in the denaturation of proteins and RNases, separating rRNA from ribosomes. Addition of chloroform forms a colorless upper aqueous phase containing RNA, an interphase containing DNA and a lower phenol-chloroform phase containing protein. RNA is collected from the upper aqueous phase by alcohol (2-propanol or ethanol) precipitation followed by rehydration.
One of the advantages of this method is the stabilization of RNA and rapid denaturation of nucleases. Besides advantages, it has several drawbacks such as it is difficult to automate, needs labor and manual intensive processing, and use of chlorinated organic reagents.
4-1.4.2. Direct lysis methods
This method involves use of lysis buffer under specified conditions for the disruption of sample and stabilization of nucleic acids. If desired, samples can also be purified from stabilized lysates. This method eliminates the need of binding and elution from solid surfaces and thus avoids bias and recovery efficiency effects.
Advantages
• Extremely fast and easy.
• Highest ability for precise RNA representation.
• Easy to work on very small samples.
• Amenable to simple automation.
Drawbacks
• Unable to perform traditional analytical methods (e.g. spectrophotometric method).
• Dilution-based (most useful with concentrated samples).
• Potential for suboptimal performance unless developed/optimized with downstream analysis.
• Potential for residual RNase activity if lysates are not handled properly.
Bibliography
Brown TA. 2006. Gene cloning and DNA analysis: an introduction. 5th ed. Blackwell Scientific.
http://amrita.vlab.co.in/?sub=3&brch=186&sim=718&cnt=1
http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/rnaisolation/general-articles/the-basics-rna-isolation.html
Reece RJ. 2003. Analysis of Genes and Genomes. John Wiley & Sons, U.K.