Each project is handled by experts and can be fully customized to suit the objectives of the project and to accommodate specific requirements. We use a personal, open and customized approach.
HTPathwaySeq generates in-depth information on drug mode of action, highlights molecular similarities between compounds and reveals potential compound-induced toxicities. Altogether, HTPathwaySeq provides critical data for candidate prioritization for further drug development.
HTTargetSeq supports the development of RNA targeting oligonucleotides such as short interfering RNAs (siRNA) and antisense oligonucleotides (ASO). HTTargetSeq addresses a key challenge in oligonucleotide drug development – accurate off-target gene identification – important for anticipation of safety issues as well as for the potential repurposing of such off-target genes as novel therapeutic targets.
Both HTPathwaySeq and HTTargetSeq are based on shallow RNA sequencing and data analysis is based on gene set enrichment analysis (HTPathwaySeq) and differential gene expression analysis paired with seed based sequence analysis (HTTargetSeq).
RNA seq is performed directly on cell lysates from 96-well culture plates and a typical experiment assesses 94 conditions plus internal controls in quadruplicate (384 samples) but depending on the project goals requirements up to 376 conditions in singlicate can be accomodated per run. To facilitate data interpretation results are shared through Savanna, Biogazelle’s proprietary visualization app.
HTPathwaySeq and HTTargetSeq enable exploitation of an unprecedented volume of data on the molecular phenotype of lead compounds, helping to make informed decisions earlier in the drug discovery process and to increase chance for success.
Biogazelle is skilled to support you with targeted gene expression analysis on a large number of samples from the different phases in your clinical trial, according to a well-defined protocol. In this context, we can measure samples for one specific gene, a limited panel of genes, or a multigene signature, whatever is required in your specific study. We support you with all information needed for sampling, sample handling and shipment. Sample processing and final reporting of the data will be done according to your requisites in a pre-defined timeframe, always to the highest quality standards.
If you need a lab with internationally recognized and accredited expertise in RT-qPCR for running your clinical samples in a routine setting, for example using a test that we developed in a previous phase for you (Lab Developed Test, LDT), Biogazelle is the perfect partner for you.
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 offers qPCR-based mRNA - miRNA - lncRNA expression analysis.
Biogazelle has a world wide expertise in the field of qPCR gained through a decade of pioneering qPCR research, making it a unique service provider for medium or large-scale gene expression studies.
We offer full customization, starting with expert consultancy to work out the optimal experiment design (including selection of stably expressed reference genes)!
The entire qPCR workflow is performed using rigorous MIQE compliant procedures (Bustin et al., Clinical Chemistry, 2009), high-throughput laboratory facilities, and state-of-the-art technologies. These high-throughput laboratory facilities enable large-scale experiments with exceptional value for money.
The final report contains reliable results generated after applying peer-reviewed approaches for quality control and data processing. The data are delivered in an easy to understand format with comprehensive graphs.
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.
Quality assurance and quality control is guaranteed during every step of the workflow and included within Biogazelle ISO 17025 accreditation scope.
qPCR gene expression data analysis is done using our own qbase+ software.
Biogazelle offers RNA sequencing workflows for different sample types, including cells, fresh frozen tissue, FFPE tissue and plasma. Starting from 100 ng of total RNA or 200 µl of plasma. In addition to gene expression analysis, transcript expression analysis, gene fusion detection, mutation analysis and allele-specific expression analysis can be conducted. Processing of RNA sequencing reads is done by Cobra, our proprietary, automated and scalable platform. Cobra incorporates multiple quality control steps along the data processing pipeline. Data is quickly processed (more than 100 samples a day), while providing data security (against data loss and security breaches).
Dedicated workflows are available depending on the sample type and the RNA molecule of interest:
Stranded polyA+ RNA sequencing
PolyA+ RNA sequencing enables expression analysis of mature polyadenylated RNAs, including the majority of all protein coding mRNAs and a subset of long non-coding RNAs. By enriching polyA+ RNA, a higher sequencing depth of these RNAs is obtained, resulting in higher sensitivity and more accurate quantification, especially of low abundant genes. A strand-specific protocol is used, which provides important information with regard to strand orientation of the transcribed RNA.
Stranded total RNA sequencing
Total RNA sequencing results in a comprehensive analysis of both coding and non-coding RNAs, regardless of polyadenylation status of the transcript, and includes all long non-coding RNAs (lncRNAs). As the entire transcriptome is considered, the most complete expression profiling is ensured. Human lncRNA annotation is based on the latest version of LNCipedia, offering one of the richest databases of human lncRNAs. Predicted functions of lncRNAs are available in our proprietary LNCarta database or can be determined using dedicated guilt-by-association studies. As with polyA+ sequencing, a strand-specific protocol is used, which provides important information with regard to strand orientation of the transcribed RNA, especially important when studying sense/antisense overlapping expression.
RNA capture sequencing
RNA capture sequencing enables the study of gene expression of low quality human samples, such as FFPE and biofluids. Sequence specific probes covering more than 98% of the human exome allow to focus on the coding regions of mRNA from these challenging samples to maximize the sensitivity and sequencing efficiency.
A subtype of the RNA capture sequencing approach is also available, targeting 1385 cancer-related transcripts, further increasing the sequencing efficiency and offering a cost-effective solution in the oncology field to study both expression levels and structural RNA changes (alternative splicing, mutations, and fusion genes).
BIogazelle has establised a 7-step RNA biomarker development program using multivariate model building enabling us to develop a robust RNA biomarker signature, based on mRNA, miRNA and/or lncRNA.
The start of our biomarker discovery program (step 1: generation of discovery data) is the comprehensive profiling of candidate RNA biomarker genes using RNA sequencing on a first patient cohort. Our platform does not only measure the 21 000 protein coding genes (messenger RNA) but also profiles non-coding genes (+2500 miRNAs and/or +60 000 long non-coding RNAs). By also measuring the non-coding RNAs, the chances of finding a robust RNA biomarker signature are maximized. RNA sequencing enables the unbiased, specific and sensitive discovery of transcripts expressed in your samples. This will allow you to investigate the expression levels of both known and unknown transcripts. Our workflow is optimized to handle clinically relevant sample types including, whole blood, formalin fixed tissues and liquid biopsies (e.g. serum, plasma, urine).
The RNA sequencing data will serve as starting point to develop a biomarker signature in a two-step approach: a feasibility study (step 2), followed by biomarker panel discovery and signature establishment (step 3).
Next, a qPCR-based biomarker assay will be developed. Indeed, while RNA sequencing is used as preferred technology for biomarker discovery, RT-qPCR constitutes a powerful platform for your clinical and diagnostic applications. To this end, a technology switch from RNA sequencing to RT-qPCR will be performed. In practice this means that for each gene of the biomarker panel identified based on the RNA sequencing data, a qPCR assay will be designed and validated. Subsequently, qPCR data will be generated on the same cohort of samples as used in phase 1 (step 4). The qPCR data will then serve to optimize the biomarker signature (step 5).
Finally, the biomarker signature will be validated in an independent cohort of samples using the RT-qPCR-based test previously developed (step 6-7).
In parallel to steps 4 to 7, our expert partner can also implement web applications enabling candidate or final models to be implemented, either for demonstration purposes or for use in research or in routine, depending on the level of validation of the results.
The end result of our biomarker discovery and validation program is a validated qPCR-based test to measure the identified RNA biomarker signature.
Biogazelle’s experienced data-analysis service unit analyses the expression data generated through our (small) RNA seq or qPCR gene expression services, using advanced analysis methods, tailored to your needs.
RNA seq data processing
Processing of RNA sequencing reads is done by Cobra, our proprietary, automated and scalable platform. Cobra processes both small RNA as well as messenger RNA and long non-coding RNA sequencing data with state-of-the-art tools. Small RNA data processing is built on proprietary code based on a long standing expertise in the field (Mestdagh et al., Nature Methods, 2014). Cobra incorporates multiple quality control steps along the data processing pipeline. Data is quickly processed (more than 100 samples a day), while providing data security (against data loss and security breaches).
RNA seq data analysis
Basic data analysis includes differential expression analysis for small RNA, coding and long non-coding genes or transcripts. In addition, we offer comprehensive and customized data analyses such as benchmark studies, complex experimental design, pathway analysis, disease subtyping, among others. A PhD level data analysis expert is part of each project team.
Dedicated resources to study non-coding RNA
Our data analysis pipelines include access to dedicated long non-coding RNA and small RNA functional annotation databases (such as decodeRNA and our proprietary LNCarta). We have generated an expanded human gene catalog and assembled a comprehensive reference transcriptome including lncRNA genes from dedicated databases such as LNCipedia and Ensembl.
LNCarta is Biogazelle’s proprietary database of predicted lncRNA functions through high-throughput perturbation by chemical compounds and silencing of transcription factors through siRNA. LNCarta assists in (1) mapping lncRNAs onto pathways, (2) providing functional context for lncRNAs, (3) and identifying upstream regulators of lncRNAs.
qbase+ is our commercially available desktop software for qPCR data-analysis on Mac and Windows computers. The user-friendly software is based on peer-reviewed quantification models for PCR efficiency correction, error propagation, inter-run calibration and statistics (Hellemans et al., Genome Biology, 2007). Advanced normalization methods and an improved geNorm algorithm for selection of stably expressed reference genes are built into the software (Vandesompele et al., Genome Biology, 2002).
PCR assay design
Best-in-class PCR assays are designed using our proprietary and ISO17025 accredited primerXL design engine, taking into account secondary structures that impact PCR efficiency, SNPs in primer annealing regions that impact robustness and accuracy, transcript diversity, and ultimate specificity by avoiding putative off-target amplification.
Small RNA sequencing enables the analysis of small non-coding RNA molecules, typically in the range of 20-40 nucleotides. The small RNA sequencing work-flow focusses on microRNAs (including isomiRs) as the most prominent class of small RNAs, by specifically size selecting the miRNA fraction using Pippin Prep technology (Sage Sciences).
Biogazelle offers dedicated small RNA seq workflows for different sample types, including cells, fresh frozen tissue, FFPE tissue and body fluids (serum, plasma, urine, etc). Starting from 100 ng of total RNA or 200 µl of body fluid.
Sequencing reads are processed using Biogazelle’s proprietary small RNA sequencing analysis pipeline Cobra and annotated according to the latest miRBase database. In addition to microRNAs, other small RNAs such as piRNAs, tRNA and sn(o)RNAs (fragments) are also quantified.
Biogazelle offers qPCR and dPCR assay design and validation.
We trust on our deep knowledge and many years of experience when developing and validating a PCR assay that fulfills the end-user requirements. If you are looking for a specific transcript or you want to get full ownership of such assays for your gene expression study, we use our proprietary and accredited primerXL design engine. Next to that, we can offer you the commercially available off-the-shelf PrimePCR assays that also meet the highest level of quality. These superior assays were expertly designed and fully wet lab validated by Biogazelle according to qPCR community driven MIQE guidelines (Bustin et al., Clinical Chemistry, 2009).
Either we run the validated assays in Biogazelle’s accredited lab on your samples of interest, or you run them in your lab, depending on your preference.
In case you have specific questions towards mutation analysis or gene copy number variation analysis, Biogazelle is able to design and validate digital PCR assays, which can then also be applied on your precious samples in our lab that acts as a European reference lab for the QX droplet digital PCR technology (Bio-Rad).
Sample-specific validated approaches for RNA extraction are applied.
RNA extraction is offered coupled to our (small) RNA seq or qPCR gene expression analysis services.
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. Biogazelle’s co-founders dr. Jan Hellemans and prof. dr. Jo Vandesomple are co-author on the digital MIQE guidelines (Huggett et al., Clinical Chemistry, 2013). Biogazelle is also serving as a reference laboratory for Bio-Rad’s QX200TM Digital Droplet PCRTM system (ddPCR) in Europe and uses his experience to provide digital PCR services, including dPCR assay design using our proprietary primerXL design engine, for academic and industrial customers. Digital PCR services are performed in an ISO17025 certified environment and following the good clinical laboratory practice (GCLP) guidelines, allowing us to analyse samples from clinical trials.
Biogazelle has already performed many ddPCR projects, both in R&D and clinical trial setting, including (i) gene copy number quantification to determine the stability of transgenic cells or organisms, (ii) splice variant quantification, (iii) quantification of pathogenic organisms, (iv) rare event detection and copy number analysis on cell free DNA and DNA extracted from FFPE tissue for cancer diagnostics and monitoring of patient response to treatment, and (v) copy number analysis for standards used in GMO analysis.
The Biogazelle diagnostics laboratory supports your digital PCR projects and will fully assist in experiment design and data-analysis. Quality assurance and quality control is guaranteed during each and every step of the workflow.
"The interaction with Biogazelle was top-notch. Communication on parameters of the study were fluid and alignment on the expectations on both sides was easy. The work was performed efficiently (quickly) and the quality control measures seem to be top notch. The data package received was clear and met our expectations. Overall this collaboration was a success on all counts."
Biogazelle has not received any endorsements.