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Are there biomarkers in the blood for the type of autism I study? QIAGEN kits can help me collect, stabilize and isolate RNA from my blood sample. I‘ll use the QIAseq RNA Library Prep Kit and then run my samples on an NGS instrument. This will show me the RNA levels in the blood sample. The Biomedical Genomics Workbench shows me that 21 key genes have markedly lower levels in autism patients than in my control patients. The IPA software shows that these genes are involved in behavioral disorders, such as autism. NGS has identified 21 genes that change in autism. Two techniques are needed to confirm a result like this, so I‘ll verify my data with qPCR. Do these 21 RNAs that I identified with NGS also change when I look at them by qPCR? I can use the same RNA that I isolated for RNAseq — it‘s best to test RNA from the same sample for consistent results. I built a custom RT 2 Profiler PCR Array with the GeneGlobe ® Custom Builder tool — this will let me look at only the RNAs that interest me. IPA tells me exactly which pathways these RNAs belong to - some are involved in cell death and others in neurological development. GeneGlobe‘s Data Analysis Portal shows me that 14 of the original 21 RNAs change in the same direction with qPCR as they do in RNAseq. These 14 RNAs could be a reliable blood biomarker signature, but I need to validate in a larger study. I‘m going to need some help with a study of this size. I‘ve gathered blood samples from 200 autism patients and 200 controls. Now I can send them to QIAGEN‘s Service Core and have them check for my 14 RNAs by qPCR to see if this biomarker signature is valid. My results from the Service Core are promising! The Biomedical Genomics Workbench indicates that the RNA signature is present in 94.6% of the autism samples. Time to see if I can get this into a clinical trial! Sample to Insight: RNA samples Sample collection stabilization isolation Targeted library construction NGS run Data analysis Interpretation QIAGEN Webinar Program: “RNA Biomarker Development: Sample to Insight“ PAXgene Blood RNA Kit miRNeasy Serum/Plasma Kit QIAseq rRNA Depletion Nano Kit RNeasy MinElute Cleanup Kit QIAseq RNA Library Prep Kit QIAseq Size Selection Kit QIAseq Library Quant System Biomedical Genomics Workbench Ingenuity Pathway Analysis Software Ingenuity Pathway Analysis Software Sample collection stabilization isolation cDNA synthesis PCR run Data analysis Interpretation QIAGEN Webinar Program: “Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR)“ “Critical Factors for Successful Real-Time Multiplex qPCR“ You can use qPCR to verify your sequencing results; RNAseq and qPCR are an increasingly common combination for discovery and verification in RNA research, including: mRNA, miRNA and lncRNA. Do the 21 miRNAs identified by RNAseq show the same changes by qPCR analysis? Biomarker Discovery via RNAseq Verification via qPCR Sample collection stabilization isolation cDNA synthesis PCR run Data analysis Interpretation QIAGEN Webinar Program: “Fundamental Concepts and Special Considerations in RNA Isolation“ “Biomarker Discovery, from Sample to Biomarker“ miRNeasy Serum/Plasma Kit You can use the same isolated RNA from your original blood samples to make sure that you‘re looking at the same RNA profile. miRNeasy captures all RNA >18 nt, giving you the flexibility to study multiple RNA species. Sample collection stabilization isolation cDNA synthesis PCR run Data analysis Interpretation RT 2 Profiler PCR Array RT 2 lncRNA PCR Array RT 2 SYBR ® Green ROX qPCR Mastermix RT 2 First Strand Kit miScript miRNA PCR Array GeneGlobe Custom Builder Tool QIAGEN provides customizable qPCR arrays for mRNA, miRNA and lncRNA – just send a list of unique RNAs that you want to test, and we send you back a pre-made array. It‘s easy with our GeneGlobe Custom Builder. Here we‘re building a custom RT 2 Profiler PCR Array for mRNA. Sample collection stabilization isolation cDNA synthesis PCR run Data analysis Interpretation QIAGEN Webinar Program: “PCR Array Data Analysis Tutorial“ GeneGlobe Data Analysis Portal Ingenuity Pathway Analysis Software You can then get insight from your qPCR array results with GeneGlobe’s Data Analysis Portal. Then, confirm with the same software you used earlier, Ingenuity Pathway Analysis, that the same pathway is affected. Here, 14 of the original RNAs are verified, and revealed further insight into their roles in cell death and neurological development. Larger Studies via QIAGEN‘s Service Core Sample collection stabilization isolation cDNA synthesis PCR run Data analysis Interpretation QIAGEN Webinar Program: “QIAGEN Service Solutions for Biomarker Research“ GeneGlobe Data Analysis Portal Again, the answer to one question leads into another. You have a biomarker signature of 14 RNAs that works well in the samples you‘ve tested. But if this signature can ever make the jump to clinical use, you need confirmation that it works in a much larger cohort. The first step is to expand your sample size and repeat the testing. If your lab isn’t big enough to handle such a large number of samples, you can get help from QIAGEN’s Service Core. Sample collection stabilization isolation cDNA synthesis PCR run Data analysis Interpretation QIAGEN Service Core When life science biomarker research leads to a promising result in a large group of samples, the next step can be to move this research into a clinical trial, where it takes its first steps toward being used for diagnosis. This is the ultimate goal of much of “translational” or “bench-to-bedside” research – to discover something that can be turned into a tool for clinicians and used in molecular diagnostics. Sample collection stabilization isolation cDNA synthesis PCR run Data analysis Interpretation QIAGEN Service Core Researchers in the life sciences face many decision points. Should you study a particular molecule? Should you use a certain technique? Here, there were multiple points at which we were required to choose a certain path. The path selected, is highlighted in blue. We could have used biofluids, cells or tissue as our starting sample. After looking at messenger RNA, we could have examined regulatory mechanisms like microRNA or long non-coding RNA, or perhaps DNA methylation. You have many avenues to explore to fully understand why a phenomenon is happening, and a full study can take years and hundreds of individual experiments to complete. Going down the wrong path could cost considerable time and money. This is where QIAGEN comes in – we help our life sciences customers at every stage of these multi-stage, multi-year studies, not only with products, but also with advice and content that can help you make the right decisions. Sample to Insight Trademarks: Ingenuity ® , GeneGlobe ® , QIAGEN ® , Sample to Insight ® , (QIAGEN Group). SYBR ® (Life Technologies Corporation), PAXgene ® (PreAnalytix GmbH). © 2016 QIAGEN, all rights reserved Sample collection stabilization isolation Targeted library construction NGS run Data analysis Interpretation Sample collection stabilization isolation Targeted library construction NGS run Data analysis Interpretation Sample collection stabilization isolation Targeted library construction NGS run Data analysis Interpretation Sample collection stabilization isolation Targeted library construction NGS run Data analysis Interpretation Every life sciences study begins with a question. For example, put yourself in the shoes of this autism researcher who is watching a webinar about biomarkers in the blood. It sparks your curiosity - could you find biomarkers like these in your study? The first step is to stabilize the blood, such as with the PAXgene ® Blood RNA Kit then isolate with the miRNeasy Serum/Plasma Kit. You might then choose to use RNA sequencing to look at all the RNAs in your sample. QIAGEN’s NGS solutions let you perform RNAseq using whatever NGS instrument you have in your facility. The QIAseq RNA Library Prep Kit generates a library of all the RNAs present in your sample. Once you‘ve collected your data, you can then generate insights from it using bioinformatics solutions like the Biomedical Genomics Workbench and Ingenuity ® Pathway Analysis. Here, 21 genes are lower in autism patients, and IPA confirms the involvement of these genes in certain behavioral disorders. Having discovered by RNA sequencing that 21 genes are altered in autism, a new question arises. How can you verify that the changes in RNA are real? Analysis with different techniques sometimes yields different results. Fortunately, QIAGEN has a solution – our qPCR portfolio. RNA profiling is a powerful technique for understanding cellular origins and disease states. Recent studies in a variety of diseases have revealed RNA signatures that are excellent biomarker candidates for understanding disease status and predicting progression. Suppose you want to discover a biomarker. What are the major steps in discovering a biomarker when you start from a blood sample? Here is the story of a researcher who is trying to find blood-based biomarkers for autism spectrum disorders. So you‘ve identified a set of genes that are differentially expressed in autism. What‘s your next move? Verification by the gold standard, quantitative RT-PCR. Let‘s explore the next steps together. Frequently, your next step in biomarker discovery will be to test your signature in a larger cohort. When you need more hands to handle such a large sample size, QIAGEN‘s Service Core can help. Our experts isolate nucleic acids, run sequencing and qPCR experiments, and even help with data analysis. Let‘s see how it works. Blood sample RNA (mRNA) Identify potential RNA biomarkers by DA Autism- focused targeted RNAseq Autism- focused qPCR array Total RNAseq Verify by a custom qPCR array qPCR verification with more samples Clinical study— diagnostic marker Targeted RNAseq DNA methylation cause mRNA differences? DNA methylation analysis by Pyroseq miRNA and lncRNA cause mRNA differences? miRNA and lncRNA analysis via RNAseq Verify levels by qPCR Verify effect on mRNA w/ functional studies DNA Identify genetic variants by DA Whole genome sequencing Targeted DNA sequencing Whole exome sequencing Verify by qPCR qPCR verification with more samples Disease- variant association study Functional studies Drug development Clinical study— diagnostic marker Life Science Researcher: Decision Tree Solid blue circles represent the workflow choices made in the above example.
