Comparison of two RNA isolation methods' for determination of SOD1 and SOD2 gene expression in human blood and mononuclear cells
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In the current study, two RNA isolation techniques were compared and their abilities to produce high-quality RNA were evaluated. mRNA expression profiles of SOD1 (Cu/Zn superoxide dismutase) and SOD2 (Mn superoxide dismutase) genes were measured by real-time PCR. From a pool of fresh human citrate-whole blood and ten healthy individuals, RNA was isolated with the TRIzol (TM) extraction method (TRI) and with the ABI PRISM (TM) 6100 Nucleic AcidPrepStation (ABI). The concentration and purity of RNA extracts were determined spectrophotometrically. RNA integrity was evaluated by electrophoresis on a 1% agarose gel. PCR was performed on a 7500 Real-Time PCR System. The student's t-test was applied to compare normally distributed variables. Both protocols gave similar RNA quantities when adjusted to the initial blood volume. Relative quantification values obtained from the TRI method for SOD1 were significantly higher (p lt 0.01) and for SOD2 were significantly lower (p lt 0.05) as compared ...to those obtained from the ABI method, respectively. Coefficients of variation (CV) for gene expression parameters in SOD1 and SOD2 analyses were lower when the TRI method was used. The TRI method was generally more consistent in yielding pure RNA in comparison to the ABI and better reproducibility in gene expression analyses was apparent.
Keywords:AbiPrism (TM) / blood / PBMC / real-time PCR / SOD / TRIzol (TM)
Source:Indian Journal of Biotechnology, 2013, 12, 4, 468-474
- Natl Inst Science Communication-Niscair, New Delhi
Funding / projects:
- Interactive role of dyslipidemia, oxidative stress and inflammation in atherosclerosis and other diseases: genetic and biochemical markers (RS-175035)
- The study was supported by COST BM904.