Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems

Abstract

Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser-capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser-capture microdissection. Arabidopsis root cells undergoing giant cell formation as a result of nematode infestation and uninfested control root cells were laser-captured and used to evaluate two amplification systems. One, NuGEN's WT-Ovation Pico (Pico) amplification system, uses total RNA as starting material, and the other, NuGEN's WT-One-Direct (One-Direct) amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The One-Direct system was less reproducible and more variable than the Pico system. The Pico amplification kit resulted in the detection of thousands of differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the One-Direct amplification kit.

Keywords: cryosectioning; differential expression; root knot nematode; technical replicates.

FIGURE 1

Experimental design. Arabidopsis plants, 3 weeks post-inoculation with juvenile M. incognita, were divided into three biological replicates (BR). Giant cells and control root cells were collected from each biological replicate. All biological replicates were processed with Pico or One-Direct kits. Biological replicates 1 and 2 were subdivided further into three technical replicates.

FIGURE 2

Differences in gene-expression levels observed after Pico and One-Direct amplifications. Kernel density estimates (A) of transcript distributions across the genome. Pico amplifications are shown in blue and One-Direct in red. Box plots (B) for each microarray. Biological replicates with technical replicates (TR) are indicated. The box ends define the 25th and 75th quantiles, and the green line identifies median values.

FIGURE 3

Correlations across microarray data sets. Principle (Prin) components analysis (A) is plotted for Pico amplifications in gold (giant cell) and blue (control cells) and One-Direct amplifications in green (giant cells) and red (control cells). A correlation heat map (B) was generated by unsupervised hierarchical clustering of samples. Colors are as in A.

FIGURE 4

Variation in reproducibility according to amplification protocol. Scatter-plots were generated using the natural log of the MAS5 signals and are shown with the associated weighted κ statistic for the indicated technical replicates. Red ovals indicate 95% density ellipse.

FIGURE 5

Comparison of gene-expression measurements after Pico and One-Direct amplification. Scatter-plot with regression line (blue) of the difference between the natural log of the giant cell MAS5 signal and the natural log of the control cell MAS5 signal for Pico (y-axis) and One-Direct (x-axis). A 45° trend (perfect agreement) line is drawn in orange.