RT2 Profiler PCR Arrays allow researchers to analyze gene expression in biological pathways or disease states using real-time PCR. The document discusses:
1. PCR Arrays focus on profiling genes relevant to specific pathways or disease states. They provide a simple, reproducible, and sensitive way to simultaneously profile expression of many genes related to a pathway.
2. Examples are provided demonstrating how PCR Arrays have been used in cancer research to discover breast cancer biomarkers, in immunology research to monitor cytokine induction, and in toxicity research to determine drug toxicity profiles.
3. Key advantages of PCR Arrays are highlighted, including their simplicity, performance, relevance to specific pathways, and ability to analyze gene expression from small amounts
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
Multicopy reference assay (MRef) — a superior normalizer of sample input in D...QIAGEN
Copy number variations (CNVs) and alterations (CNAs) are a source of genetic diversity in humans and are often pathogenic. Numerous CNVs and CNAs are being identified with various genome analysis platforms, including array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping platforms, and next-generation sequencing. Independent verification of copy number changes is a critical step. Quantitative real-time PCR (qPCR) is a classic method to verify microarray copy number findings. Traditional copy number assays that use qPCR typically rely on a putative single-copy gene reference assay (e.g., RNase P or TERT) to normalize the DNA input for downstream ΔΔCT-based copy number calculation for comparison to a reference genome. When applied to cancer samples, these single-copy reference assays may no longer be a reliable indicator of DNA input due to the presence of complex chromosome composition (both in chromosome number and structure). To meet the need for an accurate DNA input normalizer, especially for heterogeneous tumor samples, QIAGEN developed a multicopy reference (MRef) assay for real-time PCR copy number analysis. This assay, in conjunction with QIAGEN’s greater than 10 million genomewide copy number assays and pathway- and disease-focused copy number PCR arrays (Figure 1), provides a successful solution for copy number analysis. This article will address the assay design considerations, development, and performance of this multicopy reference (MRef) assay.
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
Multicopy reference assay (MRef) — a superior normalizer of sample input in D...QIAGEN
Copy number variations (CNVs) and alterations (CNAs) are a source of genetic diversity in humans and are often pathogenic. Numerous CNVs and CNAs are being identified with various genome analysis platforms, including array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping platforms, and next-generation sequencing. Independent verification of copy number changes is a critical step. Quantitative real-time PCR (qPCR) is a classic method to verify microarray copy number findings. Traditional copy number assays that use qPCR typically rely on a putative single-copy gene reference assay (e.g., RNase P or TERT) to normalize the DNA input for downstream ΔΔCT-based copy number calculation for comparison to a reference genome. When applied to cancer samples, these single-copy reference assays may no longer be a reliable indicator of DNA input due to the presence of complex chromosome composition (both in chromosome number and structure). To meet the need for an accurate DNA input normalizer, especially for heterogeneous tumor samples, QIAGEN developed a multicopy reference (MRef) assay for real-time PCR copy number analysis. This assay, in conjunction with QIAGEN’s greater than 10 million genomewide copy number assays and pathway- and disease-focused copy number PCR arrays (Figure 1), provides a successful solution for copy number analysis. This article will address the assay design considerations, development, and performance of this multicopy reference (MRef) assay.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Advanced miRNA Expression Analysis: miRNA and its Role in Human Disease Webin...QIAGEN
miRNAs are small functional RNAs, which regulate gene expression post-transcriptionally. The miScript miRNA PCR Array System is a sensitive and reliable technology for detection of mature miRNAs in any laboratory. In this slideshow, the challenges of miRNA data analysis and solutions that the miScript miRNA PCR Arrays provide for researchers interested in identifying miRNA from cells, tissues and FFPE samples are described. You will also learn how to use our GeneGlobe Data Analysis Center to identify miRNAs that may be important in your favorite biological pathway or disease.
RT2 Profiler PCR Arrays: Pathway-focused Gene Expression Profiling with qRT-P...QIAGEN
This paper evaluates the performance of the newest technique for monitoring the expression of a panel of pathway- or disease-specific genes: the RT2 Profiler PCR Array System. The RT2 Profiler PCR Array System combines the quantitative performance of SYBR® Green real-time PCR with the multiple-gene profiling capabilities of a microarray.
The RT2 Profiler PCR Array is a 96- or 384-well plate containing RT2 qPCR Primer Assays for a set of 84 related genes, plus 5 housekeeping genes and 3 controls. The complete system includes an instrument-specific master mix and an optimized first strand synthesis kit. This paper presents experimental data showing that RT2 Profiler PCR Arrays have the sensitivity, reproducibility, and specificity expected from real-time PCR techniques. As a result, this technology brings focused gene expression profiling to any biological laboratory setting with a real-time PCR instrument.
SuperScript IV Reverse Transcriptase for RNA Analysis | ESHG 2015 Poster PS14...Thermo Fisher Scientific
Survey and interview studies conducted over a three year period revealed that researchers are not satisfied with their current reverse transcriptase and are performing reactions with increasingly difficult samples, such as poorly purified RNA and unpurified RNA (direct RT) that both contain inhibitors. To meet this performance gap, the Thermo Fisher Life Sciences Solutions group produced a new reverse transcriptase, SuperScript® IV, and experiments we performed show that it is the most robust reverse transcriptase compared to other enzymes. SuperScript® IV characterization was performed in the context of “real world” situations where users do not have perfect RNA samples. In the presence of a variety of inhibitors, we demonstrate that SuperScript® IV possesses superior performance in a variety of inhibitors, such as alcohols, salts, detergents, phenol, heparin, hematin, bile salts, and formalin typically found in sample preparation reagents, cell lines, blood, feces, and FFPE samples. This enzyme can even detect RNA targets in unpurified RNA samples (directly lysed cells) and whole blood without sacrificing sensitivity and yield. The introduction of SuperScript® IV enables researchers to obtain more consistent results independent of sample quality and simplify and speed up workflows by eliminating RNA purification.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Functional analysis by mRNA knockdown using siRNAs is now routine in many molecular biology labs. However, many RNAi experiments fail due to diversion from simple, good practices. This webinar will review the steps leading to successful siRNA experiments, including:
- Understanding the target transcript
- siRNA selection
- Choosing the cell type
- Validating the assay
- Including appropriate biological controls
Deeper Insight into Transcriptomes! Download the FlyerQIAGEN
Discover a new workflow for RT-PCR-based gene expression work
Accurate and biologically relevant results in RT-PCR-based
gene expression can be difficult to achieve. Successful
transcriptome work requires validated, reproducible targets
and high-quality technology. Recognizing the variability arising from sample physiology
and pathology, the influence of sample purification and
assay conditions, and the importance of access to easyto-
use software, QIAGEN experts developed a new gene
expression workflow. It will help you properly validate your
RT-PCR and gain the deepest insight into your result.
The enzyme Telomerase maintains telomeres at the ends of
chromosomes. The Telomerase Reverse Transcriptase (TERT)
gene codes for the enzyme’s catalytic domain and is not
expressed in normal somatic cells. As a consequence, normal
cells acquire senescence by shortening of their telomeres
during cell division and eventually undergo apoptosis. In
contrast to normal somatic cells, expression of TERT is
reinstated in cancer cells causing escape from senescence and
apoptosis by maintaining the telomeres. It has recently been
shown that mutations in the TERT promoter region play a key
role in regulating and reinstating TERT expression. Up to 90%
of cancers carry a mutation in the TERT promoter region.
Mutations like C228T and C250T create new binding sites for
the E26 transformation-specific (ETS) transcription factor that
regulates TERT expression (1,2). Experimental evidence
showed that the ETS factor GA-binding protein, alpha subunit
(GABPA) binds to the de novo ETS motif and activates TERT
transcription in cancer cells.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Advanced miRNA Expression Analysis: miRNA and its Role in Human Disease Webin...QIAGEN
miRNAs are small functional RNAs, which regulate gene expression post-transcriptionally. The miScript miRNA PCR Array System is a sensitive and reliable technology for detection of mature miRNAs in any laboratory. In this slideshow, the challenges of miRNA data analysis and solutions that the miScript miRNA PCR Arrays provide for researchers interested in identifying miRNA from cells, tissues and FFPE samples are described. You will also learn how to use our GeneGlobe Data Analysis Center to identify miRNAs that may be important in your favorite biological pathway or disease.
RT2 Profiler PCR Arrays: Pathway-focused Gene Expression Profiling with qRT-P...QIAGEN
This paper evaluates the performance of the newest technique for monitoring the expression of a panel of pathway- or disease-specific genes: the RT2 Profiler PCR Array System. The RT2 Profiler PCR Array System combines the quantitative performance of SYBR® Green real-time PCR with the multiple-gene profiling capabilities of a microarray.
The RT2 Profiler PCR Array is a 96- or 384-well plate containing RT2 qPCR Primer Assays for a set of 84 related genes, plus 5 housekeeping genes and 3 controls. The complete system includes an instrument-specific master mix and an optimized first strand synthesis kit. This paper presents experimental data showing that RT2 Profiler PCR Arrays have the sensitivity, reproducibility, and specificity expected from real-time PCR techniques. As a result, this technology brings focused gene expression profiling to any biological laboratory setting with a real-time PCR instrument.
SuperScript IV Reverse Transcriptase for RNA Analysis | ESHG 2015 Poster PS14...Thermo Fisher Scientific
Survey and interview studies conducted over a three year period revealed that researchers are not satisfied with their current reverse transcriptase and are performing reactions with increasingly difficult samples, such as poorly purified RNA and unpurified RNA (direct RT) that both contain inhibitors. To meet this performance gap, the Thermo Fisher Life Sciences Solutions group produced a new reverse transcriptase, SuperScript® IV, and experiments we performed show that it is the most robust reverse transcriptase compared to other enzymes. SuperScript® IV characterization was performed in the context of “real world” situations where users do not have perfect RNA samples. In the presence of a variety of inhibitors, we demonstrate that SuperScript® IV possesses superior performance in a variety of inhibitors, such as alcohols, salts, detergents, phenol, heparin, hematin, bile salts, and formalin typically found in sample preparation reagents, cell lines, blood, feces, and FFPE samples. This enzyme can even detect RNA targets in unpurified RNA samples (directly lysed cells) and whole blood without sacrificing sensitivity and yield. The introduction of SuperScript® IV enables researchers to obtain more consistent results independent of sample quality and simplify and speed up workflows by eliminating RNA purification.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Functional analysis by mRNA knockdown using siRNAs is now routine in many molecular biology labs. However, many RNAi experiments fail due to diversion from simple, good practices. This webinar will review the steps leading to successful siRNA experiments, including:
- Understanding the target transcript
- siRNA selection
- Choosing the cell type
- Validating the assay
- Including appropriate biological controls
Deeper Insight into Transcriptomes! Download the FlyerQIAGEN
Discover a new workflow for RT-PCR-based gene expression work
Accurate and biologically relevant results in RT-PCR-based
gene expression can be difficult to achieve. Successful
transcriptome work requires validated, reproducible targets
and high-quality technology. Recognizing the variability arising from sample physiology
and pathology, the influence of sample purification and
assay conditions, and the importance of access to easyto-
use software, QIAGEN experts developed a new gene
expression workflow. It will help you properly validate your
RT-PCR and gain the deepest insight into your result.
The enzyme Telomerase maintains telomeres at the ends of
chromosomes. The Telomerase Reverse Transcriptase (TERT)
gene codes for the enzyme’s catalytic domain and is not
expressed in normal somatic cells. As a consequence, normal
cells acquire senescence by shortening of their telomeres
during cell division and eventually undergo apoptosis. In
contrast to normal somatic cells, expression of TERT is
reinstated in cancer cells causing escape from senescence and
apoptosis by maintaining the telomeres. It has recently been
shown that mutations in the TERT promoter region play a key
role in regulating and reinstating TERT expression. Up to 90%
of cancers carry a mutation in the TERT promoter region.
Mutations like C228T and C250T create new binding sites for
the E26 transformation-specific (ETS) transcription factor that
regulates TERT expression (1,2). Experimental evidence
showed that the ETS factor GA-binding protein, alpha subunit
(GABPA) binds to the de novo ETS motif and activates TERT
transcription in cancer cells.
Two-Tailed PCR - New Ultrasensitive and Ultraspecific Technique for the Quant...Kate Barlow
Mikael Kubista, Department of Biotechnology, CAS and TATAA Biocenter
We present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on novel chemistry called Two-tailed RT-qPCR. It takes advantage of target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of 7 logs and a sensitivity sufficient to detect less than ten target miRNA molecules. The reverse transcription step can be multiplexed and it allows for rapid testing with a total analysis time of less than 2.5 hours.
This slidedeck presents a simple and accurate real-time PCR system for relevant biological pathway- and disease-focused mRNA and long noncoding RNA (lncRNA) expression profiling. Learn about the stringent performance built into the technology to ensure its sensitivity, specificity, reproducibility and reliability. Application examples are also presented.
Please note: This presentation accompanies a recorded webinar at:
https://www1.gotomeeting.com/register/347794241
Biomarkers for studying gene regulation and cell function can be efficiently analyzed by multiplexed methods. Dr. Jim Lazar from OriGene Technologies will provide an overview of four different but related detection technologies that can be used to analyze genetic variants, microRNA expression, transcription factor binding, and protein expression on the Luminex xMAP platform. OriGene’s broad panel of assays and tools for discovery, analysis and validation of multiple classes of important biomarkers will allow researcher to develop more accurate descriptions of biologically complex systems.
Next generation sequencing of the whole transcriptome enables high resolution measurement of gene expression activity in different tissue and cell types. This methodology provides an in depth study of known transcripts and depending on the data analysis, allows identification of additional transcript types such as transcript variants, fusion transcripts, and small and long ncRNAs.
In this study we performed RNA-Seq using the Ion Torrent™ sequencing platform to compare the expression profile of testicular germ cell cancers (seminoma type, n=3) and normal testis (n=3). Using Partek Flow® 3.0 and TopHat/BowTie or Star aligners, we aligned the reads to the human genome and mapped sequences to the RefSeq database. Differentially expressed genes were identified and screened with additional germ cell tumors.
PCA analysis showed clear separation of the two sample types indicating biological differences. List of differentially expressed genes generated from TopHat/Bowtie and Star were similar. We identified a large number of genes that were up and down regulated with high degree of significance (p<0.01,>2X FC (fold change)). These included genes related to testicular tissue type, stem cell pluripotency (NANOG; POU5F1) and proliferation (KRAS, CCND2).
In addition, a number of differentially expressed noncoding RNAs were identified (SNORD12B, XIST). The method was validated on a small set of genes (n=20) using qPCR (TaqMan® Assays) and were found to be correlated. We used the OpenArray® platform to quickly and quantitatively screen 102 differentially expressed genes and 10 endogenous control genes across a number of different testicular germ cell cancer types.
We used a complete work flow solution from sample prep to NGS to qPCR to compare the expression profile of normal testis and seminoma type germ cell tumors. From the NGS experiments we identified a large number of differentially expressed genes for qPCR screening with samples from different types of germ cell tumors. Results from these screening studies will be presented.
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
Total RNA Discovery for RNA Biomarker Development WebinarQIAGEN
Precision medicine offers to transform patient care by targeting treatment to those with most to gain. To date the most significant advances have been at the level of DNA, for example, the use of somatic DNA alterations as diagnostic indicators of disease and for prediction of pharmacodynamic response. Development of RNA expression signatures as biomarkers has been more problematic. While RNA expression analysis has yielded valuable insights into the biological mechanisms of disease, RNA is a more unstable molecule than DNA, and more easily damaged or degraded during sample collection and isolation. In addition, RNA levels are inherently dynamic and gene expression signatures are extraordinarily complex. Recently, much progress has been made in identifying key changes in gene expression in cancer and other diseases, as well as identifying expression signatures in circulating nucleic acid that have the potential to be developed into diagnostic and prognostic indicators.
Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.
1. PCR ARRAYS
RT2 ProfilerTM PCR ARRAYS
Apoptosis
Cancer
Cell Adhesion
Cell Signaling
Cytokines
Disease
Inflammation
Neuroscience
Stem Cells
Toxicology
Focus on your Pathway
2. PCR ARRAYS: Pathway Analysis with Real-Time PCR
How PCR ARRAYS Work
Focus on Your Pathway
What are PCR Arrays?
RT² Profiler PCR Arrays are the most reliable and sensitive gene
expression profiling technology for analyzing a focused panel of
genes in signal transduction, biological process, or disease related
pathways using real-time PCR.
How are PCR Arrays Utilized?
The RT² Profiler PCR Arrays have been increasingly used in
research on cancer, immunology, stem cells, toxicology, biomarker
discovery and validation, and phenotypic analysis of cells and
transgenic animals.
1. Convert Total RNA to cDNA.
cDNA 1
cDNA 2
2. Add cDNA to RT2 qPCR Master Mix & Aliquot Mixture Across
PCR Array.
Why PCR Arrays?
Simplicity
1.E-000
1.E-000
Delta Rn
1.E+001
1.E-001
1.E-003
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
10-6
1.E-01
10-4
1.E-02
10-3
10-2
CCL8
CCR1
CCR2
CCR8
CCR9 CEBPB
CCR3
CCR4
CCR5
CCR6
CCR7
CCL5
CCL7
CRP
CX3CR1 CXCL1 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL2 CXCL3 CXCL5 CXCL6 CXCL9
ICEBERG IFNA2
IL10
IL10RA IL10RB
IL13 IL13RA1 IL17C
IL1A
IL1B
IL1F10
IL1F5
IL8
IL8RA
IL8RB
IL1F6
IL1F7
IL1F8
IL1F9
IL1R1
IL1RN
IL22
IL5
IL5RA
IL9
IL9R
LTA
LTB
LTB4R
MIF
SCYE1
SPP1
TNF
HGDC
RTC
RTC
RTC
B2M
HPRT1 RPL13A GAPDH ACTB
HOUSEKEEPING
GENE CONTROLS
GENOMIC REVERSE TRANSCRIPTION
CONTROLS
DNA CONTROL
TNFSF5 TOLLIP XCR1
PPC
PPC
PPC
POSITIVE
PCR CONTROLS
4
Breast Tumor
Complete Systems Available
RT² First Strand Kit: cDNA Synthesis
Eliminates Genomic DNA
Insures RT Efficiency & Consistency
20-Minute Protocol: Quick & Simple
SYBR Green qPCR Master Mixes
Specific Amplification - No Primer Dimers
Maximum Amplification Efficiencies
Formulated for the Instrument in Your Lab
Data Analysis: Convert Raw Ct Data to Fold-Change Results
Easy-to-Use Web Portal
http://www.SABiosciences.com/pcrarraydataanalysis.php
Each well in a PCR Array represents the expression of a gene
related to a pathway or disease state. RTC Controls require the
use of the RT2 First Strand Kit.
www.SABiosciences.com
1.E-06
D
3
1.E+00
CCL4
CCL11 CCL13 CCL15 CCL16 CCL17 CCL18
-2
-1
0
1
2
Fold Change Ratio (log2)
TIMP3
1.E-01
CCL3
CCL1
C
-3
MMP9
ITGA2
MCAM
ITGB4
1.E-02
CCL20 CCL21 CCL23 CCL24 CCL25 CCL26
C5
1.E-04
ITGB3
1.E-03
CCL2
C4A
TGFB1
1.E-03
1.E-05
10-1
-4
CCNE1
CDKN2A
FGFR2
1.E-04
CCL19
C3
1.E+00
A
B
A
10-5
1.E-05
BCL6
Cycle Number
1.E-06
ABCF1
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
4. Data Analysis.
D
Human Inflammatory Cytokines & Receptors RT2 Profiler PCR Array
1.E-003
Cycle Number
1
Anatomy of a PCR ARRAY
1.E-001
1.E-002
1.E-002
Normal Breast
Relevance
PCR Arrays focus on profiling the genes relevant to the
pathways or disease states of your interest.
Profile 2
Profile 1
1.E+001
Delta Rn
Performance
The PCR Arrays have the sensitive, reproducible, specific,
and reliable peformance to accurately profile multiple genes
simultaneously in 96- or 384-well formats.
3. Run in Your Real-Time PCR Instrument.
p-Value for Fold Change
The simplicity of the PCR Arrays makes expression profiling
accessible for routine use in every research laboratory with
a real-time PCR instrument.
Support@SABiosciences.com
3. PCR ARRAYS: Performance
Reproducibility
Sensitivity
100
High Reproducibility Among Different Users
Use as little as 25 ng Total RNA
B1
40
B2
40
B3
40
A1
20
20
20
20
10
10
10
0
0
0
10
20
30
40 0
40
10
20
30
40 0
40
10
20
30
0
40 0
40
30
20
500
1000
100
Input RNA [ng]
50
30
20
20
20
10
0
40
0
10
20
30
40 0
40
10
20
30
40 0
40
10
20
30
0
40 0
40
30
30
20
20
20
10
0
0
40
0
10
20
30
40 0
40
10
20
30
40 0
40
10
20
30
0
40 0
40
30
A4
30
30
20
20
20
40
20
Signal [-d(RFU)/dT]
0
110
90
70
50
30
10
-10
110
90
70
50
30
10
-10
110
90
70
50
30
10
-10
70
60
80
90
BMP3
9.8
08
70
60
BMP5
70
60
80
80
BMP7
60
90
8.8
22
90
8.
72
70
BMP1
80
BMP2
90
BMP3
130
110
90
70
50
30
10
-10
110
90
70
50
30
10
-10
130
110
90
70
50
30
10
-10
110
90
70
50
30
10
-10
10
30
40
10
20
30
40
10
0
10
20
30
40
0
0
10
20
30
40
0
0
10
20
30
40
0
0
PCR Arrays Yield Highly Reproducible Results
Four replicate sets of raw threshold data (1-4) obtained by two
different scientists (A & B) at two different times on Human Drug
Metabolism RT2 Profiler PCR Arrays
Correlation Coefficients
are directly compared. The results
A4
A2
A3
A1
R2
demonstrate a high degree of corB1 0.993 0.989 0.995 0.992
2
relation (R > 0.990).
0.992
80
70
60
Accuracy
8.8
97
BMP2
BMP4
90
8.5
43
120
Uniformly High PCR Amplification Efficiency
100
80
70
60
80
70
60
90
8.7
38
BMP6
90
BMP15
8.7
61
80
70
60
Tm [Co]
BMP4
20
Ct from User B
Amplification Efficiency [%]
8.
86
10
20
10
Single Dissociation Curves
BMP1
40
B2 0.994 0.990 0.995
B3 0.992 0.990 0.993 0.992
B4 0.993 0.992 0.994 0.992
Specificity
80
30
30
0
60
20
10
0
10
100
10
20
10
25
PCR Arrays Let You See More Genes with Less RNA
Different amounts of universal total RNA were characterized
using the Human Inflammatory Cytokines and Receptors PCR
Array, and the percentage of detectable genes was calculated for each RNA amount.
As little as 25 ng total RNA yields greater than an 80% positive
call, even for cytokines expressed at very low levels.
40
30
10
A3
30
Human Inflammatory Cytokines & Receptors PCR Array
30
10
0
20
20
10
10
30
0
A2
30
10
Ct from User A
% Positive Calls
30
40
40
0
30
0
60
30
10
80
B4
40
30
BMP5
BMP6
BMP7
90
BMP15
Agarose Gel
PCR Arrays Amplify A Single Gene-Specific Product in Every Reaction
Universal total RNA was characterized on the TGFβ / BMP Signaling
Pathway PCR Array, followed by dissociation (melt) curve analysis.
PCR Arrays specifically detect individual genes despite the expression
of related gene family members in the same RNA sample.
80
60
40
20
0
Gene
PCR Arrays Yield the Most Accurate Results
A representative set of assays for 4,000 genes used in RT2 PCR
Arrays have an average amplification efficiency of 99% with a
95% confidence interval from 90-110%.
Consistently high amplification efficiencies enable PCR
Arrays to accurately analyze multiple genes simultaneously
utilizing the ΔΔCt method.
4. PCR ARRAYS: Application Examples
Cancer Research: ECM PCR ARRAYS for Breast Cancer Biomarker Discovery
ECM & Cell Adhesion
101
UP
-R
EG
UL
AT
ED
Breast Tumor
100
10-1
C L A
O 4 2
C N D
T N 2
S L
E E
10-2
A A T 1
D M S
I G 4
T B
M P
M 9
T M 3
I P
10-3
M P
M 3
10-5
C T 1
N N
F 1
N
DO
WN
-R
EG
UL
AT
ED
I G 3
T B
10-4
Extracellular Matrix &
Adhesion Signaling Pathway
ECM & Cell Adhesion PCR Arrays Revealed
Up- & Down-Regulated Genes in Breast Cancer
Total RNA from normal human breast and a
human breast tumor were characterized in
technical triplicates, and the relative expression
levels for each gene in the two samples are
plotted against each other in the Scatter Plot.
Genes encoding the matrix metallopeptidases
(MMP3 & MMP9) and their inhibitors (TIMP3) are
up-regulated, while genes encoding integrins
(ITGB3 & ITGB4) are down-regulated, by at least
three-fold (outside the silver field) in breast
tumors relative to normal tissue.
NO
10-5
GE
AN
CH
10-3
10-4
10-2
101
100
10-1
Normal Breast
Immunology Research: Monitoring Cytokine Induction with PCR ARRAYS
Common Cytokines
10-9
10-8
I 9
L
10
-7
C F
S 2
I 1
L 0
I 1
L A
T F
N
I 1
L B
10-6
p value
S A I T C L S G I I A C
T T S I A
I N F C N E
Common Cytokine PCR Array Identified 23 Up-Regulated
and 6 Down-Regulated Genes Following PMA and
Ionomycin Treatment
Triplicate total RNA samples from human peripheral
blood mononuclear cells (either untreated or stimulated
with 50 ng/mL PMA and 1 µg/mL ionomycin for 6 hours)
were characterized.
Twenty-three cytokine genes are up-regulated (> 5-fold,
p < 0.0005) including interleukins, colony stimulating
factors, and TNF ligands after 6 hours of stimulation. Six
interleukin and TNF ligand genes are down-regulated
under the same conditions.
T F F 0
N S 1
L A
T
10
I 1
L 1
C F
S 1
I 5
L
-5
P G A
D F
T F 2
G B
10-4
I 2
L 1
I 2
L 2
I 1
L 3
I 3
L
I 1
L 7
T F F 1
N S 1
T F F 3
N S 1 B
B P
M 6
I N 5
F A
10-3
I 2
L
I N
F G
T F S S 1 B
N R P F 1
T F F 4
N S 1
B P
M 3
I 1 7
L F
10-2
10-1
10 0
-7 -5 -3 -1
1 3 5 7 9 11 13 15 17
fold difference [log2]
Common Cytokines Network
Toxicity Research: Determining Drug Toxicity with PCR ARRAYS
128
FAX:
119
30
20
888.465.9859
INTERNATIONAL TEL:
HSPA6
CRYAB
CSF2
MT2A
HSPA1A
TNF
DDIT3
HSPH1
CYP1A1
HSPA5
HSPCA
DNAJB4
HMOx1
10
1GADD45A
DNA Repair
Mechanism Pathway
888.503.3187
5005
Actos
Avandia
Rezulin
125
0
USA TEL:
Stress & Toxicity PathwayFindertm
18SrRNA
1ACTB
Fold Up-Regulation
5000
Stress & Toxicity PathwayFinder PCR Array
Uncovered Distinct Gene Expression Profiles
Associated with Liver Toxicity Caused by 3
PPARγ Agonists
RNA from HepG2 cells treated with three different
glitazone PPARγ agonists for type 2 diabetes
mellitus was characterized, and the results were
compared to that of a vehicle (DMSO) control.
A withdrawn drug with idiosyncratic liver toxicity
(Rezulin) induces very different changes in the
expression of stress-related genes than two safer
drugs still on the market (Avandia and Actos).
+1 301.682.9200
FAX:
+1 301.682.7300
5. What’s Your Pathway?
Pathway / Topic Focus
Alzheimer’s Disease
Angiogenesis
Angiogenic Growth Factors & Angiogenesis Inhibitors
Apoptosis
Breast Cancer and Estrogen Receptor Signaling
Cancer Drug Resistance and Metabolism
Cancer miRNA
Cancer PathwayFinder
Cell Cycle
Chemokines and Receptors
Common Cytokines
DNA Damage Signaling Pathway
Drug Metabolism
Drug Transporters
EGF / PDGF Signaling Pathway
Extracellular Matrix and Adhesion Molecules
Growth Factors
Heat Shock Proteins
HIV Infection & Host Response
Hypoxia Signaling Pathway
Inflammatory Cytokines and Receptors
Inflammatory Response & Autoimmunity
Innate & Adaptive Immune Responses
Interferons (IFN) and Receptors
JAK / STAT Signaling Pathway
MAP Kinase Signaling Pathway
Neurogenesis & Neural Stem Cells
Neuroscience Ion Channels and Transporters
Neurotransmitter Receptors and Regulators
Neurotrophins and Receptors
NF B Signaling Pathway
Nitric Oxide Signaling Pathway
Notch Signaling Pathway
Osteogenesis
Oxidative Stress and Antioxidant Defense
p53 Signaling Pathway
PI3K / AKT Signaling Pathway
Signal Transduction PathwayFinder
Stem Cell
Stress and Toxicity PathwayFinder
TGFβ / BMP Signaling Pathway
Th17 for Autoimmunity and Inflammation
Th1-Th2-Th3
Toll-Like Receptor Signaling Pathway
Tumor Metastasis
Tumor Necrosis Factor (TNF) Ligands and Receptors
Wnt Signaling Pathway
Custom PCR Arrays
PCR Array Gene Expression Analysis Service
Which RealTime PCR Instrument?
PCR ARRAY Catalog #
Rat
Human Mouse
PAHS-057
PAHS-024
PAHS-072
PAHS-012
PAHS-005
PAHS-004
MAH-102
PAHS-033
PAHS-020
PAHS-022
PAHS-021
PAHS-029
PAHS-002
PAHS-070
PAHS-040
PAHS-013
PAHS-041
PAHS-076
PAHS-051
PAHS-032
PAHS-011
PAHS-077
PAHS-052
PAHS-064
PAHS-039
PAHS-061
PAHS-404
PAHS-036
PAHS-060
PAHS-031
PAHS-025
PAHS-062
PAHS-059
PAHS-026
PAHS-065
PAHS-027
PAHS-058
PAHS-014
PAHS-405
PAHS-003
PAHS-035
PAHS-073
PAHS-034
PAHS-018
PAHS-028
PAHS-063
PAHS-043
Inquire
Inquire
PAMM-057
PAMM-024
PAMM-072
PAMM-012
PAMM-005
PAMM-004
MAM-102
PAMM-033
PAMM-020
PAMM-022
PAMM-021
PAMM-029
PAMM-002
PAMM-070
PAMM-040
PAMM-013
PAMM-041
PAMM-076
PAMM-051
PAMM-032
PAMM-011
PAMM-077
PAMM-052
PAMM-064
PAMM-039
PAMM-061
PAMM-404
PAMM-036
PAMM-060
PAMM-031
PAMM-025
PAMM-062
PAMM-059
PAMM-026
PAMM-065
PAMM-027
PAMM-058
PAMM-014
PAMM-405
PAMM-003
PAMM-035
PAMM-073
PAMM-034
PAMM-018
PAMM-028
PAMM-063
PAMM-043
Inquire
Inquire
PARN-057
PARN-024
PARN-072
PARN-012
PARN-005
PARN-004
PARN-033
PARN-020
PARN-022
PARN-021
PARN-029
PARN-002
PARN-070
PARN-040
PARN-013
PARN-041
PARN-076
PARN-051
PARN-032
PARN-011
PARN-077
PARN-052
PARN-064
PARN-039
PARN-061
PARN-404
PARN-036
PARN-060
PARN-031
PARN-025
PARN-062
PARN-059
PARN-026
PARN-065
PARN-027
PARN-058
PARN-014
PARN-405
PARN-003
PARN-035
PARN-073
PARN-034
PARN-018
PARN-028
PARN-063
PARN-043
Inquire
Inquire
Supported PCR Instruments
Applied Biosystems (ABI)
7000, 7300, 7500, 7500 FAST, 7700, 7900HT,
StepOnePlus (96- & 384-well blocks)
Bio-Rad
iCycler, iQ5, MyiQ, Chromo4, Opticon, Opticon 2
Stratagene
Mx3000P, Mx3005P, Mx4000
Roche
LightCycler 480 (96- & 384-well bocks)
Eppendorf
Mastercycler ep realplex
Complete the PCR ARRAY System
Product
Pack Size
Price
The RT2 First Strand Kit is required for use with PCR Arrays.
RT2 First
Strand Kit
12 Arrays
$ 199
PCR Array pack sizes of 12 & 24 offer the best value.
PCR Arrays
2 Arrays (96-well)
12 Arrays (96-well)
24 Arrays (96-well)
4 Arrays (384-well)
$ 499
$ 1599
$ 2499
$ 999
RT2 SYBR Green qPCR Master Mixes are necessary for use
with PCR Arrays. The Master Mix is available with ROX,
Fluorescein, or SYBR Green only.
Master
Mix
2 Arrays
12 Arrays
24 Arrays
$ 189
$ 999
$ 1599
Guaranteed Performance
Over 100 Pathways Available
Focus on your Pathway