RNA preparation of Saccharomyces cerevisiae using the digestion method may give misleading results

Abstract

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.

Fig. 1

Gene expression profiles following zymolyase treatment. FC fold change, con non-treated control, zym 300 U/ml for 10 min

Fig. 2

Gene expression changes induced by RNA polymerase. RNA polymerase-coding genes, which indicated significant changes (FC > 2.0), were extracted. The gene expressions of non-treated control samples were adjusted 1-fold. FC fold change

Fig. 3

Gene expression changes in cell wall genes. Cell wall protein-coding genes which indicated significant changes (FC > 2.0) were extracted (a). To enable comparisons with previous research, the gene expression of SLT2 was measured by semi-quantitative PCR (b). FC fold change, con non-treated control, Zym 300 U/ml for 10 min, Zym 2h 4 U/ml for 2 h. Bars = SE. n = 3

Fig. 4

Gene expression changes in the MAPK pathway. MAPK pathway genes (FC > 1.0, p < 0.05) were extracted and classified to each pathway. The root structure of MAPK was constructed by referring to KEGG [20]. Lined box, induced gene; dashed line box, repressed gene. MAPKKK MAP kinase kinase kinase, MAPKK MAP kinase kinase, MAPK MAP kinase

Fig. 5

Oxidative stress responses. Oxidative stress response genes (FC > 1.0, p < 0.05) were extracted and classified to each localization (a). FC fold change. SOD expressions and SOD activity (b). Dashed line graph, SOD1 expression; line graph, SOD2 expression; gray bar graph, SOD activity. con non-treated control, Zym 300 U/ml for 10 min, Zym 2h 4 U/ml for 2 h. Bars = SE. n = 3