Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor genes

Abstract

Direct labeling of RNA is an expedient method for labeling large quantities (e.g. micrograms) of target RNA for microarray analysis. We have developed an efficient labeling system that uses T4 RNA ligase to attach a 3'-biotinylated donor molecule to target RNA. Microarray analyses indicate that directly labeled RNA is uniformly labeled, has higher signal intensity than comparable labeling methods and achieves high transcript detection sensitivity. The labeled donor molecule we have developed allows the attachment of multiple biotins, which increases target signal intensity up to 30%. We have used this direct-labeling method to detect previously discovered class predictor genes for two types of cancer: acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In order to test the sensitivity of direct RNA labeling, we analyzed the AML and ALL expression profiles for predictor genes that were previously found to show elevated expression in the disease state. Direct labeling of AML poly(A) RNA detects 90% of the class predictor genes that are detected by the IVT-based target amplification method used to discover the genes. These results indicate that the detection sensitivity, simplicity (single tube reaction) and speed (2 h) of this direct labeling protocol may be ideal for diagnostic applications that do not require target amplification.

Figure 1

Structure of nucleotide donor molecule pCpB₃. Multiple biotin labels attached to the donor 3′-hydroxyl are separated by HEG and TEG spacers. Concatenation of at least five biotins to the donor molecule can be accomplished without significantly inhibiting ligation efficiency.

Figure 2

Condensation reaction of adenosine-5′-monophosphoromorpholidate and 5′pCp(TEG-biotin)-3′.

Figure 3

Gel shift analysis of RNA 20mer labeling. The first lane (MW) shows 100 bp Ladder (NEB); the second lane (RNA) contains the 20 nt model RNA substrate before ligation and the third lane (LIG) after ligation to pCpB. Lane +SA contains ligated RNA incubated with streptavidin. Image analysis of lanes 3 and 4 indicate that >95% of the RNA substrate is labeled and shifted. The appearance of two bands in the shifted lane is likely caused by variation in the number of subunits in the streptavidin holoenzyme.

Figure 4

Gel shift analysis of direct-labeled RNA and internally labeled cRNA. RNA 20mer direct-labeled with pCpB (lane 1) and incubated with streptavidin to affect a gel shift (lane 2). Internally labeled cRNA (1 μg) was fragmented by magnesium hydrolysis (lane 3) and incubated with streptavidin (lane 4). Unlabeled cRNA (500 ng) was fragmented by magnesium hydrolysis and direct-labeled with pCpB (lane 5) and incubated with streptavidin (lane 6). Note that under the same ligation conditions, 95% of the RNA 20mer is labeled and 55% of the magnesium hydrolyzed cRNA is direct labeled. Of the internally labeled cRNA fragments, 75% contain one or more biotin molecule as evidenced by multiple bands in the streptavidin-shifted lane (4).

Figure 5

Comparison of cRNA fragmented by RNase III and magnesium hydrolysis. Unlabeled cRNA fragmented with RNase III (RN) has a similar size distribution to cRNA fragmented by magnesium hydrolysis (Mg). Fragments 20–100 nt are ideal for array hybridization.

Figure 6

Gel shift assay of cRNA fragmented with RNase III and direct-labeled with pCpB. After RNase III fragmentation and labeling (RN), 93% of the cRNA fragments are shifted when incubated with streptavidin (+SA).

Figure 7

Array performance of direct-labeled (DL) cRNA and internally labeled (IL) cRNA. Triplicate cRNA samples were prepared from total heart RNA and hybridized to U133A arrays under standard conditions (45°C).

Figure 8

Array performance of cRNA direct-labeled with donor molecules containing multiple biotins. Average signal is increased by labeling with multiple biotins and detection sensitivity (%P) is improved.