Transcribed-ultra conserved region expression profiling from low-input total RNA

Abstract

Background: Ultra Conserved Regions (UCRs) are a class of 481 noncoding sequences located in both intra- and inter-genic regions of the genome. The recent findings that they are significantly altered in adult chronic lymphocytic leukemias, carcinomas, and pediatric neuroblastomas lead to the hypothesis that UCRs may play a role in tumorigenesis.

Results: We present a novel application of Ribo-SPIA isothermal linear amplification of minute RNA quantities for quantifying Transcribed-UCR (T-UCR) expression by quantitative PCR. Direct comparison of non-amplified with amplified cDNA in two neuroblastoma cell lines showed that the amplification approach increases sensitivity and repeatability in T-UCR quantification. It is noteworthy that the Ribo-SPIA step allowed us to analyze all 481 T-UCRs by using 150 ng of RNA, while introducing a minimal bias and preserving the magnitude of relative expression. Only the less abundant T-UCRs have high intra-assay variability, consistently with the Poisson distribution statistics and stochastic effects on PCR repeatability.

Conclusions: We demonstrated that the quantification procedure shown here is an accurate and reliable technique for genome-wide non coding gene (i.e., UCRs) profiling using small amounts of RNA. This issue is particularly important because studies of transcription regulation are increasingly conducted in small homogeneous samples, such as laser capture microdissected or sorted cell populations.

Figure 1

Plots showing the relation between the average Cq value and the standard deviation for T-UCRs in LAN-5 cell line, using reverse transcriptase of total RNA (A) and amplification system (B). Bar plots display the mean SD value for T-UCRs with an average Cq value ranging between 15-20, >20-25, >25-30, and >30-35 cycles.

Figure 2

Scatter plots showing the linear correlation (r) between normalized Cq values obtained with the SPIA™ system (dCq_A) and by reverse transcriptase of total RNA (dCq_NA) in LAN-5 (A) and GI-ME-N (B) cell lines.

Figure 3

Plot of the difference in differential T-UCR expression between LAN-5 and GI-ME-N cell lines against each mean dCq_NA value. Bar plot shows the mean |ΔΔCq| value for T-UCRs with an average Cq_NA value ranging between 15-20, >20-25, >25-30, and >30-35 cycles.