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Open Access Methodology article

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

Paola Scaruffi1*, Sara Stigliani1, Simona Coco1, Franscesca Valdora2, Carla De Vecchi1, Stefano Bonassi3 and Gian Paolo Tonini1

Author Affiliations

1 Translational Paediatric Oncology, National Cancer Research Institute (IST), Largo R Benzi 10, Genoa, 16132, Italy

2 Department of Oncology and Genetics (DOBIG), University of Genoa, Largo R Benzi 10, Genoa, 16132, Italy

3 Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via della Pisana 235, Roma, 00163, Italy

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BMC Genomics 2010, 11:149  doi:10.1186/1471-2164-11-149

Published: 3 March 2010

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.