Thursday, 28 November 2013
ISSS Abstract: Application of probabilistic functional integrated network analysis to the study of autoimmunity and primary Sjögren's Syndrome
ISSS Abstract: The United Kingdom Primary Sjögren's Syndrome Registry (UKPSSR), a valuable resource for future pSS research
Wednesday, 27 November 2013
ISSS Abstract: Proteomic analysis of pooled blood serum samples to detect symptom-specific changes in primary Sjögren's Syndrome patients
ISSS Abstract: Integration of gene expression data with functional interaction and annotation data reveals patterns of connection between pSS-associated genes and the cellular processes in which they are involved
Thursday, 21 November 2013
ISSS Abstract: Effects of globin mRNA on whole blood gene expression signature in primary Sjögren’s syndrome
Al-Ali S, Cockell S, Tarn JR, James K, Young DA, the UKPSSR study group, Griffiths B, Bowman S, Ng WF
Objectives : Gene expression profiling using peripheral whole blood is a useful approach to identify biomarkers in primary Sjögren's syndrome (PSS). However, using whole blood comes with challenges as a result of the presence of globin mRNA, which represent approximately 70% of the total RNA, potentially reducing the accuracy and sensitivity of microarray analysis. The aim of this study was to evaluate the effect of globin mRNA on whole blood gene expression profiling in PSS .
Methods: Twenty-four peripheral whole blood samples from PSS patients and healthy controls (HC) were used (PSS=12, HC=12) , RNA was extracted according to the PAXgene Blood RNA kit protocol, followed by purification and concentration using the RNeasy MiniElute kit. For each sample, half of the RNA underwent a globin mRNA removal step using the GLOBINclear kit (the "globin-clear group") with the remaining half representing the "globin-present group". The efficiency of globin mRNA clearance was assessed by the ratio of real-time RT-PCR of β-globin mRNA between paired "globin-present" and "globin-clear" samples. Illumina microarrays were performed for all samples and the data analyzed using Genespring GX (Agilent).
Results: b-globin gene expression in 'globin-clear' samples was markedly reduced compared the paired 'globin-present' samples (fold change=139-555) indicating efficient removal of globin mRNA. Consistently, an RNA quality check of the globin-clear samples using a Bioanalyser confirmed lack of a sharp peak of 700 bp which represent the globin mRNA. The Probe intensity signals of the microarray were higher and the quality of the microarray data improved in the 'globin-clear' group. Independent analysis of the 'globin-clear' and the 'globin-present' groups identified 15995 versus 15173 transcripts/entities detectably expressed (1.2-fold p>0.05) between PSS and healthy controls respectively, suggestive of an improvement in the sensitivity of the data after globin-removal. When focussing on those most highly differentially expressed genes (based on relative fold changes), there was significant concurrence between the two methods. Despite the small sample size, the differentially expressed genes identified in this study were similar to a previous report.
Conclusion: Removal of globin mRNA enhanced the quality of the samples and the microarray intensity as well as enhance sensitivity, although there were considerable overlap between the differentially expressed genes identified regardless of whether globin mRNA was removed especially for the genes with high relative levels of expression between PSS and controls.
Friday, 15 November 2013
Tarn JR, Cockell S, Gillespie C, Al-Ali S, James K, the UK primary Sjögren's syndrome registry, Griffiths B, Bowman S, Young DA, Ng WF
Background: Micro RNAs (miRNAs) are 18-25nt non-coding RNAs that bind target/complementary sequences within the 3'UTR of RNA molecules steering them towards degradation or translational repression, and play a key role in the regulation of gene expression. Better understanding of the expression pattern of miRNAs in diseases may improve our understanding of the biological basis of the disease and identify potential biomarkers. The role of miRNAs in primary Sjögren's syndrome (PSS) and PSS-related lymphoma remains poorly understood. The aim of this project is to identify a miRNA signature for PSS-related lymphoma.
Methods: We profiled the expression of miRNAs in whole blood (PaxGene) total RNA preparations using the Exiqon miRCURY LNA array which encompasses >1400 miRNAs and other small non-coding RNAs. A discovery cohort comprised of 36 samples (12 PSS patients with lymphoma, 12 PSS patients without lymphoma, 12 healthy controls) were used in the initial analysis. We also explored different normalisation strategies and developed an approach which we considered most appropriate for analysing these data. Real-Time PCR was used to validate the most highly differentially expressed miRNAs between groups. A miRNA signature for PSS-related lymphoma was identified using cluster analysis followed by validation with a second independent cohort of 36 patients and controls.
Results: The initial miRNA array profiling revealed a clear clustering of the 3 subject groups. Between the 'High Function' and 'Lymphoma' patient groups, 44 miRNAs were found to be differentially expressed. The differential expressions of these miRNA were validated by RT-PCR in 3 out of 9 miRNAs with the highest fold-changes between the two groups. Indeed, based on the expression levels of these 3 miRNAs were sufficient to distinguish PSS patients with lymphoma from those without. Two out of these 3 miRNAs were also differentially expressed between the same two groups in the validation cohort.
Conclusion: We have identified 2 miRNAs that are differentially expressed in peripheral blood between PSS patients with lymphoma and those without lymphoma. Identifying the mRNA targets of these miRNAs in PSS may improve our understanding of the pathogenesis of PSS-related lymphoma. Furthermore, miRNAs may be useful biomarkers for PSS-related lymphoma.