Data Management Biobanking

[Data Management and Biobanking] Services using state-of-art technologies

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26 Apr 2017
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qPCR Real-time quantitative PCR (qPCR) is one of the most widely used laboratory techniques for fast and cost-efficient quantification of nucleic acid sequences. qPCR has a superior specificity, linear dynamic range of quantification and a high level of flexibility making it the gold standard for accurate and sensitive __EXPRESSION__ profiling. “Biogazelle's founders have more than 10 years of experience in qPCR experiment design, assay development and data analysis” The Biogazelle service laboratory uses qPCR for a wide range of applications •medium and large scale __EXPRESSION__ studies of mRNA, miRNA and lncRNA targets •medium and large scale SNP or mutation genotyping projects •medium and large scale gene copy number screening For each study, we carefully consider the entire qPCR workflow starting from experiment design and preparation phase, over assay design, validation, reaction setup and actual qPCR cycling to data-analysis and reporting. For these type of applications, we typically use our 384-well qPCR instruments; if needed, we use higher throughput qPCR instruments. Quality assurance and quality control is guaranteed during every step of the workflow. All qPCR studies are performed according to MIQE compliant procedures (Bustin et al., Clinical Chemistry, 2009). Data analysis is done using our own qbase+ software. Small RNA seq Custom lncRNA microarray Biogazelle is offering small RNA sequencing as a service for microRNA gene __EXPRESSION__ profiling. While we see the value of RNA sequencing for mRNA and lncRNA, we feel the technology is currently not cost-efficient for large scale use of this method.Many workflows incorporate poly-A+ selection of RNA (either physically or through oligo-dT reverse transcription) when a substantial fraction of lncRNAs are poly-A-. Further, several workflows do not incorporate the direction of RNA transcription, which is crucial in order to discriminate anti-sense lncRNAs. Finally, as lncRNAs are on average much lower abundant than mRNA, the number of reads in typical RNA seq experiment is not sufficient to obtain a relevant capture of the lncRNA transcriptome.Summarizing, for lncRNA profiling, one needs directional, non-poly-A+ selected (i.e. true total RNA that is ribosomal RNA depleted), high coverage RNA sequencing (120-180 million reads per sample, or more). Our current microarray is therefore a much more cost-efficient platform for lncRNA profiling. The latest version of our lncRNA microarray is based on LNCipedia 2.1 that contains 32,183 lncRNA transcripts belonging to 17,512 lncRNA genes. Genes are defined by grouping transcripts in same orientation with at least one partially overlapping exon. The sources used to feed LNCipedia are LncRNAdb (september 2011 - 82 transcripts), Broad Institute (Human Body Map lincRNAs - 14,279 transcripts), Ensembl release 64 (9,069 transcripts) and Gencode 13 (19,812 transcripts). There is at least one probe for 95% of all lncRNA genes and 90% of all lncRNA transcripts available in LNCipedia. Our microarray also contains probes for all human protein genes. The advantages of our microarray:- most comprehensive lncRNA content available- suitable for simultaneous lncRNA and mRNA gene __EXPRESSION__ profiling- each probe is linked to the LNCipedia database for rich annotation of the lncRNA- only 100 ng of total RNA is needed; protocol available for working when lower input amounts if required- carefully selected probes that are highly transcript specific- more sensitive than standard RNA-sequencing Digital PCR Digital PCR is a nucleic acid quantification method with unprecedented resolution, relative sensitivity and accuracy. It allows answering the question of how much nucleic acid target molecules are present in a particular sample. In principle, any study that requires higher resolution than you can possibly get by using qPCR (ability to measure smaller differences) or higher relative sensitivity (ability to accurately quantify a minority variant in a background of wild-type sequences) could benefit from digital PCR. At Biogazelle, we have access to QX100 droplet digital PCR technology. “Biogazelle is at the forefront of digital PCR based research as it was one of the first European laboratories that had access to the technology.” Applications for digital PCR: •Gene copy number variation analysis Due to its higher resolution compared to qPCR, digital PCR certainly has advantages for accurate discrimination between higher copy numbers (for instance to distinguish 6 from 7 copies). Also for regular gene copy number quantification (1 versus 2 versus 3 copies), it appears to be superior. •Rare event detection Rare event detection is useful in the field of cancer gene mutations (diagnosis, monitoring therapy response, early relapse detection), infectious disease (pathogen detection), genetically modified organism quantification in food, and prenatal diagnosis of genetic diseases. •Gene __EXPRESSION__ analysis Applications where higher relative sensitivity is required (e.g. ultra low __EXPRESSION__), higher resolution (smaller __EXPRESSION__ differences) or absolute quantification (e.g. splice variants), digital PCR is the methodof choice. •Clinical diagnostics Data interpretation is straightforward and results can easily be shared and interpreted among different users because absolute concentrations are determined. Moreover, its inherent high precision, accuracy and relative sensitivity are relevant features for laboratory test that are used in the clinic. Biogazelle is the European ddPCR reference lab: Biogazelle is at the forefront of digital PCR based research as it was one of the first European laboratories that had access to the technology. Furthermore, Biogazelle has an agreement with Bio-Rad to act as a reference center for the QX100 digital droplet PCR (ddPCR) system in Europe. This is completely in line with the Biogazelle mission to address unmet needs through excellence in science and technology. The biogazelle service laboratory supports your ddPCR projects and will fully assist in experiment design up to the data-analysis. Quality assurance and quality control is guaranteed during each and every step of the workflow. At any time, our experts welcome the opportunity to discuss the needs of your project. DMAS qPCR Double-mismatch allele specific (DMAS) qPCR is a cost-effective sequence variant (SNP, mutation) genotyping method based on 2 parallel qPCR reactions using SYBR Green I and modified allele specific primers (Lefever et al., in preparation). Biogazelle has in-house expertise to design and validate DMAS-qPCR assays, run the genotyping study on its 384-well qPCR instruments, and to determine the genotypes using proprietary algorithms and software. Advantages: •cost-effective alternative for probe based qPCR genotyping •high design and lab validation success rate •use of synthetic templates to demonstrate accurate results on homozygous and heterozygous genotypes •sensitivity typically down to 5% (mutant allele in background of wild-type) •available assays for common SNPs and clinically relevant mutations in cancer (BRAF, EGFR, KRAS)

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