Main

Microarray-based transcription profiling studies have provided important advances in our understanding of differential gene expression in heterogeneous cell populations—for example, in cancerous versus normal tissue. The Illumina Expression BeadChips, a popular choice for array-based expression profiling, use biotin-labeled aRNA (sometimes called complementary RNA, or cRNA) as the target. We evaluated BeadChip array results obtained using biotin-aRNA produced by two Epicentre kits: the TargetAmp-Nano and the TargetAmp-Pico Target Labeling Kits for Illumina Expression BeadChip.

High yields of biotin-aRNA

The TargetAmp-Nano and TargetAmp-Pico kits use an improved Eberwine1 linear RNA amplification and labeling procedure that generates microgram amounts of biotin-aRNA from nanogram and picogram amounts of total RNA, respectively. The kits have been optimized to maximize the yield of biotin-aRNA and to minimize reaction time and the number of pipetting steps. A TargetAmp-Nano Kit reaction is completed in about 6 h, which enables the user to prepare biotin-aRNA target and begin BeadChip hybridization on the same day.

Table 1 reports the yield of biotin-aRNA produced from a TargetAmp-Nano kit reaction and from a TargetAmp-Pico kit reaction using various amounts of input total RNA. The actual yield of biotin-aRNA produced from a sample is dependent on the poly(A) RNA content of the total RNA sample, the amount of input RNA and the integrity of the RNA. Both the TargetAmp-Nano and TargetAmp-Pico kits produce sufficient biotin-aRNA for sample replicates or for archiving.

Table 1 Yield of biotin-aRNA from the TargetAmp-Nano and TargetAmp-Pico kits.
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Genes detected

One of the most important metrics used to evaluate a microarray target labeling method is the number of genes detected by the labeled target. We prepared biotin-aRNA from 100 ng of mouse liver and mouse skeletal muscle RNA with the TargetAmp-Nano kit, and from 50 pg and 100 pg of mouse liver and mouse muscle total RNA with the TargetAmp-Pico kit. The labeled targets were hybridized to the Mouse Ref-8 Expression BeadChip array (Illumina). In a separate experiment, we prepared biotin-aRNA from 500 ng each of RNA extracted from human ovary and testicle using the TargetAmp-Nano kit. The labeled targets were hybridized to the HumanHT-12 v4 Expression BeadChip array (Illumina). The data generated were normalized using BeadStudio (Illumina). The number of genes detected (P < 0.05) from each sample is shown in Figure 1. The large number of genes detected, even with biotin-aRNA produced from as little as 50 pg of RNA, demonstrates the robust target labeling provided by the TargetAmp kits.

Figure 1: Genes detected using labeled target prepared by the TargetAmp-Nano and TargetAmp-Pico kits.
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Target RNA from the indicated source was labeled using either the TargetAmp-Nano or TargetAmp Pico kit. The target RNA was hybridized to either the Mouse Ref-8 Expression BeadChip or the HumanHT-12 v4 Expression BeadChip (Illumina).

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Correlation of expression ratios

We also examined the correlation of gene expression ratios using biotin-aRNA produced by the TargetAmp-Nano and TargetAmp-Pico kits. Biotin-aRNA was generated from 100 pg (TargetAmp-Pico kit) and 100 ng (TargetAmp-Nano kit) of RNA extracted from mouse liver and skeletal muscle tissue. There was a strong correlation (R = 0.93) between results obtained by the two procedures (Fig. 2), even though there was a 1,000-fold difference in the amount of RNA used for target preparation.

Figure 2: Correlation of expression ratios.
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Mouse liver/skeletal muscle expression ratios were obtained with biotin-aRNA produced from 100 ng and 100 pg of total RNA using the TargetAmp-Nano and TargetAmp-Pico kits, respectively.

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Conclusions

The TargetAmp-Nano Target Labeling Kit for Illumina Expression BeadChip and the TargetAmp-Pico Target Labeling Kit for Illumina Expression BeadChip kits generate microgram amounts of biotin-aRNA from as little as 50 pg of total cellular RNA. The biotin-aRNA produced detects a large number of genes on the Illumina Expression BeadChip arrays. Expression ratios determined with biotin-aRNA prepared with the TargetAmp-Nano and TargetAmp-Pico kits show high correlation with each other.