Various methods and kits are available for the extraction of RNA from cells, blood, tissue, and plasma. In the present study, 3 different commercially available kits for the isolation of RNA from equine whole blood based on different strategies were compared. For Tempus™ blood RNA tube with RNA preparation station, whole blood was directly lysed in Tempus™ blood RNA tube containing lysis buffer, and RNA was extracted from lysed whole blood solution. For the other two methods, WBC were separated from plasma and red RBC before extracting RNA.
High quality RNA is critical for downstream application, and the quality depends upon the technique that was employed for RNA isolation (Fleige and Pfaffl 2006). Quality of total RNA was assessed on the basis of RNA intactness and purity; 4 methods were used for assessment: (1) spectrophotometry, (2) micro-capillary chip's electrophoresis with fluorescent detection (Bioanalyzer, Agilent Technologies), (3) RNA denaturing gel electrophoresis and (4) real-time PCR.
The intactness of RNA extract was assessed by analyzing 18S and 28S subunits of ribosomal RNA on micro-capillary chip electrophoresis with fluorescent detection and on ethidium stained agarose denaturing gel electrophoresis (Strand et al. 2007 Schroeder et al. 2006 and Dumur et al. 2004), while purity of RNA was determined by calculating the ratio of A260nm/A280nm using spectrophotometer (Glasel 1995). Purity of RNA was considered adequate if the ratio was > 1.8 (Manchester 1996). In most cases, RNA samples assessed by these 3 methods would be recommended for use in downstream molecular analysis. In the present comparative study, the 3 methods above were not adequate for the assessment of RNA quality. Although, there was no DNA contamination using RNA agarose denaturing gel (Figure 2), considering the limit and sensitivity of agarose denaturing gel and the absence of Dnase treatment of the RNA extracts using TRIzol® reagent, a forth method, the relative IL-6 expression level in different RNA extracts was compared using real-time PCR.
The primer pair for IL-6 was from the same exon. If there were DNA contamination, the apparent IL-6 expression level would be falsely higher than the actual level. Real-time PCR results suggested that there was DNA contamination in the RNA extracts using the TRIzol® reagent method (Figure 3). Treating contaminated RNA extract with Dnase, the expression level of IL-6 was reduced. This observation further confirmed that trace DNA contamination occurred in the RNA isolate when the TRIzol® reagent method was used.
Some downstream applications do not tolerate any trace of DNA contamination, therefore the RNA isolates should be assessed by real-time PCR to determine whether or not trace DNA contamination has occurred, which is an index for demonstrating the quality and purity of the RNA extraction.
Comparing the RNA yield from the 3 methods, TRIzol® reagent method produced the highest, whereas the yield from the other two methods were equivalent but lower. For the TRIzol® reagent method WBC were separated from whole blood and almost all the WBC were recovered. For Tempus™ method, whole blood was lysed in their blood RNA tubes. The lysate contained high concentrations of components, and another cycle of RNA precipitation and dissolution was performed. All these factors will contribute to the reduction in RNA yield. For LeukoLOCK™ total RNA isolation method, WBC were separated from whole blood by filtration. The waste tubes containing plasma and RBC were centrifuged at 2000 × g for 10 min, and WBC layer was observed in the waste tube. The WBC in the waste tubes indicated that not all WBC could be captured by the filter. The present procedure resulted in decreased RNA yield.
High concentrations of globin transcripts in blood can confound the accurate assessment of the expression levels of genes harvested from blood (Wright et al. 2008). Abundant globin mRNA represents up to 70% of the total expressed transcripts and consequently limits the accurate detection of genes expressed at low concentrations. Thus, globin reduction is often considered a necessary step in the evaluation of whole-blood gene expression profiles via microarray assay. It has been shown that reduction of abundant globin mRNA is essential for revealing unique patterns of gene expression (Winn et al. 2010). Globin mRNA exists in reticulocytes (Guesella et al. 1979). LeukoLOCK™ total RNA isolation method removes plasma, RBC and reticulocytes, hence RNA recovered contains only minor quantity of globin mRNA. For this reason, when used in microarray assay, globin mRNA depletion procedure is not necessary. For RNA samples extracted using the other two methods, the globin mRNA depletion must be conducted before microarray assay is performed.
In this study, gene expression profiling with microarray was evaluated and validated using real-time PCR. Comparing the features of the three methods described, LeukoLOCK™ total RNA isolation system method was the best choice.