Desenvolvimento e validação de um protocolo de extração de RNA de células em cultura livre de fenol

Abstract

Ribonucleic acid (RNA) is a polymer of ribonucleotides found within prokaryotic and eukaryotic cells and it’s almost always related to gene expression. To get trustworthy results in gene expression research, extract a high purity and integrity RNA is necessary. Many techniques to isolate a high-quality RNA has been developed and improved and the most utilized nowadays are silica chromatography columns present in commercial kits and guanidinium thiocyanate - phenol – chloroform extraction technique, used in reagents such as Trizol marketed by Thermofisher or TRI Reagent marketed by Sigma - Aldrich, among others. Both techniques isolate a high purity and quality RNA. However, organic solvents like phenol are very toxic and interfere negatively in RNA quantification by absorbing UV in the same wavelength than RNA. Chromatography columns limits RNA extraction to the amount of each column can support and become an expensive method in experiments with a large number of samples. The objective of this work was developing an inexpensive protocol for a high-quality RNA isolation from cultured cells dispensing the use of organic system phenol-chloroform. RNA was isolated from k562 cultured cells (between 1 and 3 x 106 cells per reaction) in RPMI 1640 medium following standard procedure. Cells were separated from the culture medium by centrifugation and resuspended in a buffer solution (pH 7.8) containing 140 mM NaCl, 1.5 mM MgCl 2, 10 mM TRIS. The cells were fractionated in 1.5 mL reaction tubes and lysed by adding 0.2 volumes of 2 M DTT and 0.1 volume of NP-40 solution (10% v / v) and vortexing. The nuclei were removed by centrifugation and the supernatant containing the RNA was subjected to protein denaturation with guanidine thiocyanate at the final concentration of 1 M. The purification of RNA was performed by salting out with ammonium sulfate at the final concentration of 1 M. The RNA was precipitated with 0.1 volume of sodium acetate and 0.6 volumes of isopropanol at -20 ° C. The precipitated was washed with 50% isopropanol and eluted in ultrapure water. As control, RNA was extracted by columns with commercial kit following manufacturer's instructions. Extractions were performed on the same day with the same number of cells under the same conditions. Non-denaturing agarose gel Electrophoresis, Bradford, RT-PCR and qRT-PCR analyses were performed to evaluate the RNA extracted by the TGSA (guanidine thiocyanate - ammonium sulfate, described in this work) in comparison to RNA extracted from the chromatographic column. The analysis showed that the TGSA method allows the extraction of high-quality RNA with characteristics similar to those presented by the RNA extracted with chromatographic columns. In addition, required reagents budget for RNA isolation with TGSA method was made and a comparison of prices with commercial kits also. The TGSA method cost R$ 1.61 per reaction with reagents from several suppliers and R $ 2.66 per reaction with all reagents from Sigma Aldrich. This represents a value approximately 8.4 times lower than that required to perform the extraction of high quality RNA with the cheaper method (PureLink RNA mini kit - Thermo Fishier).