A Comparison of Silica Membrane-Based and Acidic Phenol Methods in RNA Isolation: Determination of the Most Effective Acidic Phenol RNA Isolation Protocol

摘要

Objective: The aim of this study is to determine the most effective method in isolating intact RNA from peripheral blood samples. Moreover, optimization of the best method's conditions is another aim, in order to reach and determine the best protocol in RNA isolation from peripheral blood.Subjects and Methods: This study was conducted during the period from April 2014- December 2015 in the central laboratory for research, faculty of medicine, Tishreen University. 1.5ml of peripheral blood samples were coljected from 30 healthy visitors to Tishreen University hospital, Latakia, Syria, after their agreement, gathered on EDTA anticoagulative tubes. Two common methods in RNA isolating were compared in this study in order to determine the most effective one, the first method depends on silica membrane principle using a commercial RNA isolating kit, and the second one is the traditional acidic phenol RNA isolating method. Optimization of the best method's conditions was also carried out, all that was in the purpose of determination of the best intact RNA isplation protocol.Results: The results have revealed that the traditional acidic phenol RNA isolation method was more effective in isolating intact RNA compared to using the silica membrane-based commercial kit. Moreover, the best acidic phenol RNA isolation conditions were also determined as follows; fresh peripheral blood samples (no more than 15 minutes after collecting the samples), EDTA 0.1M solution as red blood cells lysing solution, white blood cells lysing buffer contained Guanidine thiocyanate salts as well as a manually prepared white blood cells lysing buffer formula contained SDS 2%, the acidic degree of the phenol was 4.4 (pH= 4.4), the ratio of the organic mixture was (Acidic Phenol 125: chloroform 24: Isoamyl alcohol 1), using 4+ working micro centrifuge, washing the RNA isolated pellet twice using 75% cold ethanol, the centrifugation speed after washing the RNA pellet was 4000rpm, and solving the RNA pellet in nuclease free water.Conclusion: This study has indicated that in order to get intact RNA molecules from peripheral blood samples the acidic phenol method was the best one. Optimization of some conditions in the protocol has helped in improving the results.