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Censored Regression Models for the Analysis of Differential Gene Expression in qPCR Experiments.
Peter Pipelers, PhD Student - Teaching Assistant, Ghent University, Belgium
The detection of differential gene expression is an important application of a reverse transcription quantitative PCR experiment. Many normalization strategies and quantification models exist to ensure more accurate quantification and to remove the non-biological variation. However, there is no universal approach for dealing with undetermined Cq values.
We present an innovative unified censored linear regression approach for differential expression analysis in a qPCR setting. The method unifies the data preprocessing with the hypothesis testing for differential expression. The regression model includes the preprocessing by either extending the global mean expression normalization strategy or the usage of multiple reference genes. The undetermined values are handled as observations censored at the limit of detection.
We demonstrate that our normalization procedure is adequately stable and that the hypothesis tests for differential expression and the estimators of the log fold change are robust in the presence of undetermined values.
Quality Control of RNA Using The Agilent 2200 TapeStation System
Arunkumar Padmanaban, Application Scientist, Agilent Technologies
High quality RNA is critical for the success of downstream experiments. We present here the performance of the Agilent 2200 TapeStation system in assessing RNA sample quality. Twenty one different eukaryotic RNA samples extracted from various tissue sources with varying quality was analysed, including six serially heat degraded RNA. The samples were analysed using the R6K ScreenTape (R6K) and the High Sensitivity R6K ScreenTape (HS R6K) assays. The performance was benchmarked against the industry standard - Agilent 2100 Bioanalyzer. The TapeStation system indicates RNA quality by RINe (RIN equivalent) with value between 1 and 10, where 10 is the highest quality RNA. A positive correlation between both systems with R2 of 0.9878 for the RNA 6000 Nano and R6K assays and R2 value of 0.9474 for the RNA 6000 Pico and HS R6K assays was obtained. The RINe reported shows <4% deviation from RIN for the R6K assay and <7% for the HS R6K assay. The 2200 TapeStation system shows greater reproducibility (RINe) with CV <3% compared to <6% in the 2100 Bioanalyzer. Automated system of high throughput capabilities up to 96 samples with constant cost per sample makes the 2200 TapeStation system an ideal platform for RNA quality assessment.
A LNA™ based RT-qPCR system for discovery of a blood microRNA signature for colorectal cancer and for validation of mRNA targets
Ditte Andreasen, Scientific Manager, Exiqon A/S
microRNAs represent the most well described class of small RNAs (21-23nt) shown to function as post-transcriptional regulators of gene expression. microRNA are potentially attractive biomarkers as only a limited number of genes exist, primarily organ specific, they appear highly stable in biological samples like plasma and serum, and altered microRNA expression has already been associated with a number of diseases, including human cancers.
We have constructed miRNome qPCR arrays – the miRCURY LNA™ Universal RT microRNA PCR platform - and applied this to identify a specific microRNA signature in plasma samples from colorectal cancer (CRC) stage 2 patients, and from matched healthy controls. A large number of plasma samples have been screened using both miRNome and focussed custom microRNA qPCR panels. Our results show that early detection of CRC using a minimally invasive clinically viable approach is feasible.
The detection of the relevant microRNAs in the CRC diagnostic signature is enabled by the miRCURY LNA™ Universal RT microRNA PCR platform, which facilitates sensitive and accurate microRNA expression profiling. The vast majority of assays are able to detect down to 5-10 microRNA copies in the PCR without pre-amplification. Full profiling of all microRNAs in plasma/serum requires just 20µL of serum/plasma, while using the platform for full miRNome profiling requires as little as 40ng total RNA, or a 10µM FFPE section.
We have now developed further on the reverse transcription step of the LNA platform to enable concurrent generation of cDNA from both microRNA and other RNA species including mRNA and ncRNA. The reverse transcription provides the required cDNA in a one-tube reaction and this has allowed us to construct a microRNA-mRNA qPCR profiling panel focussing on key microRNA and mRNAs relevant for the P53 pathway. Preliminary data will be discussed from a study aiming to validate candidate microRNA targets identified in functional analysis screen of cells transfected with a LNA based miRNome inhibitor library. The concept of being able to profile both microRNA and mRNA at the same time will also be applicable to validate candidate microRNA targets identified by other methods such as arrays, next generation sequencing etc.
ChIP-qPCR and qbasePLUS jointly identify a MYCN-activated miRNA cluster in cancer
Jan Helemanns, , Biogazelle
Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) is very well suited to assess and quantify direct binding of specific regulatory proteins to genomic DNA sequences. Unfortunately, data-normalization and accurate quantification appear to be a major challenge for many users.
Here, we demonstrate that ChIP-qPCR in combination with state-of-the-art real-time PCR data-analysis software enables convenient and reliable quantification. We applied ChIP-qPCR to assess binding of transcription factor MYCN to miRNA cluster 17-92, to a positive control target, MDM2, and to a negative control target region. ChIP-qPCR was performed in two MYCN-overexpressing neuroblastoma cell lines (IMR5 and WAC2) using SYBR Green I detection chemistry in a 384-well plate and signals were normalized based on the average abundance of three non-specific genomic regions in the ChIP samples using the qbasePLUS multiple reference gene normalization technology. Fold enrichment was calculated relative to the input sample (non-precipitated) and compared to that of a fourth non-specific region (negative control target). Using this approach we were able to demonstrate strong MYCN-binding to the positive control and the miR-17-92 cluster. In keeping with this, the expression level of the miR-17-92 cluster is substantially increased in primary neuroblastoma tumor samples, in which the MYCN gene is amplified and overexpressed.
The results confirm the power of ChIP-qPCR in combination with the data-analysis software qbasePLUS to study gene regulation.
FlexScript™ LDA: Multiplexed Measurement of Gene Expression on the High-Throughput
Jan Van Gils, Director Sales & Marketing EMEA & India, Luminex Corporation
FlexScript™ LDA: Multiplexed Measurement of Gene Expression on the High-Throughput Luminex® Platform Using Ligation-dependent Amplification
Matt Grow, Thom Chang, Emily N. Kroutter, Bhavna Choudhury, Cristen Graham, Joshua A. Luthy, Ramin Saberi, Cora Lahey, & Edward A. Sekinger
Here we describe an approach that is simple, flexible, and with the throughput required for screening studies. We will demonstrate the following advantages of the FlexScript LDA assay:
• Multiplexing Flexibility: Detection of 2 to 500 transcripts from 1 sample well
• High-throughput Capabilities: Process 1,000s of samples per day on the Luminex FLEXMAP 3D®
• Sensitive performance: Accurate with inputs as low as 1,000 cells or 20 ng total RNA
• Accuracy: Data correlated to real-time PCR
Automated Liquid Handling of the New 384-Well FlexScript™ LDA Multiplex Gene Expression Solution
Jan Van Gils, Director Sales & Marketing EMEA & India, Luminex Corporation
Automated Liquid Handling of the New 384-Well FlexScript™ LDA Multiplex Gene Expression Kit
Josh Luthy, Bhavna Choudhury, Cora Lahey, Ramin Saberi, Thom K. Chang, Christine Valle
It is critical for high-throughput research assays to be easily adaptable to automation thereby enabling the user to benefit from time and labor cost reductions while maintaining quality. In this study, we will demonstrate:
• Automated liquid handling of FlexScript LDA 384 gene expression assay
• Reproducible results with high correlation to manual processing
• Faster Processing Time with 96 head pipetting
Advanced copy number variant analysis with qbasePLUS 2.0
Jan Helemanns, , Biogazelle
Copy number changes are known to be involved in numerous genetic disorders. Moreover, copy number polymorphisms of various sizes are thought to contribute to normal phenotypic variation and susceptibility to multifactorial disease.
In this context, qPCR-based copy number screening may serve as the method of choice for targeted copy number screening as it has many advantages over alternative methods, such as its low consumable and instrumentation costs, fast turnaround and assay development time, high sensitivity and open format (independent of a single supplier).
Here, we present several pilot experiments in which we performed targeted deletion screening in patients with human genetic disorders. Accurate copy number calling and objective interpretation was performed with an advanced module for copy number analysis integrated within qbasePLUS 2.0. The software allows the selection of more than one reference sample for accurate copy number calling. In addition, it provides flexibility with regard to the reference samples as these samples may have varying copy numbers. The identified copy numbers changes are visualized on a per sample basis and conditional bar colouring is applied for easy detection of deletions and amplifications.
In summary, we provide guidelines for qPCR-based copy number screening and subsequent data-analysis to improve the quality and reliability of your results.
Simultaneous measurement of 1718 long intergenic non-coding RNAs
Jan Helemanns, , Biogazelle
Long non-coding RNAs (lncRNAs) represent an important class of regulatory transcribed elements. Research involving these lncRNAs is rapidly emerging in the field of cancer research, because of their implications in important cellular processes, through new modes of action. lncRNAs most likely constitute a novel class of powerful biomarkers and expression profiling of this underexplored class of RNAs may lead to disease specific gene signatures. The lack of a high-throughput platform to reliably quantify lincRNAs has hampered their study so far. On top of that, lincRNA are generally low abundant, which is why there was a genuine need for a platform with ultimate sensitivity superseding microarray analysis. To accommodate this need, Biogazelle has developed a qPCR-based platform that allows high-throughput screening of 1718 lncRNAs per sample. Assays have been designed and successfully validated for 1666 human intergenic lncRNAs (lincRNAs) and 52 human lncRNAs. All assays underwent thorough in silico quality control, followed by extensive empirical validation according to international qPCR standards. All qPCR reactions are performed in triplicate using Wafergen’s SmartCycler platform. The power of the newly developed platform has been successfully demonstrated in several proof-of-concept studies. One of these studies involved lincRNA expression profiling in a TP53 gene perturbation model using the anti-cancer nutlin-3 compound in neuroblastoma cells. This study revealed numerous lncRNAs under the control of TP53. Recently, the newly developed platform was also used in a large regulatory network discovery study using 3-way integration of high-dimensional mRNA, miRNA and lncRNA expression data from the entire NCI60 cancer cell line panel. The data offer numerous opportunities towards a better understanding of complex regulatory networks in cancer. In conclusion, Biogazelle has developed a high-throughput, low-volume qPCR platform for the quantitative detection of lincRNAs. Biogazelle now offers lincRNA profiling in service, which offers a unique way to investigate the expression patterns of lncRNAs in health and disease.
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