ABSTRACT
Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches. Considering the diverse technologies now available for transcriptome analysis, methods for standardising measurements between platforms will be paramount if their diagnostic impact is to be maximised. Therefore, the use of RNA standards and other reference materials is also discussed.
Subject(s)
High-Throughput Nucleotide Sequencing/standards , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Animals , Biomarkers/metabolism , DNA Cleavage , Gene Library , Humans , Microfluidic Analytical Techniques , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Reference StandardsABSTRACT
BACKGROUND: Microarray data interpretation can be affected by sample RNA integrity. The ScreenTape Degradation Value (SDV) is a novel RNA integrity metric specific to the ScreenTape(R) platform (Lab901). To characterise the performance of the ScreenTape(R) platform for RNA analysis and determine the robustness of the SDV metric, a panel of intentionally degraded RNA samples was prepared. These samples were used to evaluate the ScreenTape(R) platform against an alternative approach for measuring RNA integrity (Agilent Bioanalyzer RIN value). The samples were also subjected to microarray analysis and the resulting data correlated to the RNA integrity metrics. FINDINGS: Measurement of SDV for a panel of intentionally degraded RNA samples ranged from 0 for intact RNA to 37 for degraded RNA, with corresponding RIN values ranging from 10 to 4 for the same set of samples. SDV and RIN scales both demonstrated comparable discrimination between differently treated samples (RIN 10 to 7, SDV 0 to 15), with the SDV exhibiting better discrimination at higher degradation levels. Increasing SDV values correlated with a decrease in microarray sample labelling efficiency and an increase in numbers of differentially expressed genes. CONCLUSIONS: The ScreenTape(R) platform is comparable to the Bioanalyzer platform in terms of reproducibility and discrimination between different levels of RNA degradation. The robust nature of the SDV metric qualifies it as an alternative metric for RNA sample quality control, and a useful predictor of downstream microarray performance.
ABSTRACT
Neuroblastoma is the most common extracranial solid malignancy in children. The disease possesses a broad range of clinical phenotypes with widely varying prognoses. Numerous studies have sought to identify the associated genetic abnormalities in the tumour, resulting in the identification of useful prognostic markers. In particular, the presence of multiple copies of the MYCN oncogene (referred to as MYCN amplification) has been found to confer a poor prognosis. However, the molecular pathways involved are as yet poorly defined. Metabolite profiles generated by in vitro (1)H MRS provide a means of investigating the downstream metabolic consequences of genetic alterations and can identify potential targets for new agents. Thirteen neuroblastoma cell lines possessing multiple genetic alterations were investigated; seven were MYCN amplified and six MYCN non-amplified. In vitro magic angle spinning (1)H MRS was performed on cell suspensions, and the spectra analysed to obtain metabolite concentration ratios relative to total choline (tCho). A principal component analysis using these concentration ratios showed that MYCN-amplified and non-amplified cell lines form separate classes according to their metabolite profiles. Phosphocholine/tCho and taurine/tCho were found to be significantly raised (p < 0.05) and glycerophosphocholine/tCho significantly reduced (p < 0.05) in the MYCN-amplified compared with the MYCN non-amplified cell lines (two-tailed t test). (1)H MRS of the SH-EP1 cell line and an isogenic cell line transfected with the MYCN oncogene also showed that MYCN oncogene over-expression causes alterations in phosphocholine, glycerophosphocholine and taurine concentrations. Molecular pathways of choline and taurine metabolism are potential targets for new agents tailored to MYCN-amplified neuroblastoma.
