Introduction: Extraction of RNA is a key step in reliable gene-expression profiling. A quick, proficient, and cost-effective technique for the isolation of RNA from blood is required for evaluating an expansive number of samples. Our point was to isolate RNA economically, quickly, and with at least of solvents. Therefore, we assessed distinctive RNA extraction techniques applied to frozen blood and effectively adjusted the TRIzol reagent procedure.
Methods: In this method, white blood cells haven’t been separated from the red blood cells. In the beginning, 4 ml chilled RNXplus reagent had been directly added to 1 ml frozen whole blood before it was melted and incubated for 5 min at room temperature, followed by supplementation with 1 ml chloroform. After vigorous mixing (because vortexing may expand DNA contamination of your RNA sample) and centrifugation at 12000 g for 15 min at 4°C, the upper layer was transferred to a new tube. An aliquot of 2.5 ml isopropanol was included and the subsequent mixture incubated for 30 min at room temperature, followed by centrifugation at 12000 g for 15 min to pellet RNA. The pellet was washed with 5 ml 75% ethanol and air-dried. RNA was dissolved in 40 ?l RNase-free water. Quality of extracted RNA was compared with results of the routine TRIzol method through an agarose gel and gene-expression analysis.
Results: The quality of isolated RNA was monitored by absorbance ratio. It was 1.91, exhibiting the absence of protein contaminants. The size of isolated RNA by this modified procedure was analyzed on an agarose gel. The two bans of isolates indicate that this procedure yields high-quality RNA.
Conclusion: The results of this examination shows that there are practical contrasts between RNA extraction procedures that should be considered while choosing extraction methods to be used for isolating RNA assigned for gene-expression analysis.
Keywords: RNA extraction, Gene expression analysis, TRIzol reagent procedure