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| Questions & answers |
| What are the advantages of the topic-defined PIQOR™ Microarrays? |
Convenient and cost-effective gene expression analysis
Instead of analyzing thousands of genes in parallel, PIQOR™ Microarrays enable researchers to cost-effectively analyze the gene expression profile of thoroughly selected sets of genes. Each topic-defined PIQOR Microarray targets at a particular field of research such as immunology, toxicology, cell death, stem cells, dermatology, and oncology.
PIQOR Microarrays have been designed in cooperation with dedicated experts from well-known universities and clinical partners. Based on the PIQOR Collection and supported by the bioinformatics team at Miltenyi Biotec, custom PIQOR Microarrays can be produced.
Unique bioinformatics strategy
A major benefit of PIQOR Technology is the high-quality, sequence-verified PIQOR Collection, including more than 25,000 well-documented genes for the species human, mouse, and rat:
• The homogeneous length of PIQOR fragments is 200–400 bp, leading to efficient hybridization reactions and comparable hybridization kinetics
• Corresponding orthologous fragments are generated from human, murine and rat genes, allowing a direct cross-species comparison of expression profiles
• PIQOR fragments are designed to contain no repetitive elements; thereby, preventing non-specific hybridization
• PIQOR Fragments cover all known alternative polyadenylation variants
Precise production of PIQOR Microarrays
The high-throughput arrayer, set up in a clean room facility, enables accurate production of high-quality microarrays. During the spotting of the PIQOR fragments in quadruplicates, a quality-control system monitors the position and quality of every spot online and documents all steps of microarray design and production.
If time and resources are restricted, Miltenyi Biotec provides comprehensive microarray and bioinformatics services – from RNA preparation to data analysis.
PIQOR Microarray design
• PIQORTM fragments are spotted in quadruplicates on each microarray, allowing reliable statistical analysis of gene expression data.
• Microarrays are spotted with a Piezo-based spotting unit of a high-througput arrayer. The non-contact spotting technology leads to 100% presence of the fragments on every microarray.
• Each micro-array contains six housekeeping genes and six controls, thereby improving the quantification of differential expression patterns. Genes are spotted in quadruplicates.
• PIQOR Microarray Kits* include hybridization and washing buffers, control RNAs and primer mixes for fluorescent labeling, and a suitable software package.
• Convenient protocols cover the complete microarray experiment with or without amplification, depending on the amount of starting material.
*PIQOR Microarray Kits are available in Europe only. |
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| How much RNA is required for a gene expression analysis with PIQOR™ Microarrays? |
Not only the amount but also the quality of the RNA sample is a critical factor for microarray experiments. The yield of intact mRNA from biological samples may vary between cell and tissue type, origin and status.* To generate a fluorescently labeled cDNA that allows a sensitive gene expression analysis, we recommend using the following amounts of RNA per sample.
* Submersion of fresh tissue or cell samples in an RNA-protective reagent such as PrepProtect™ Stabilization Buffer keeps the RNA intact.
If the amount of starting material is limited, such as from rare cells or biopsy material, we strongly recommend an mRNA amplification step before labeling. To speed up the amplification procedure, the µMACS One-step T7 Template Kit has been developed. Thus, microarray sample is prepared within one day: starting from magnetic mRNA isolation to in-column amplification.
When using PIQOR™ Microarrays or the Microarray Service it is possible to start with as few as 5x104 cells or 250 ng total RNA without losing much sensitivity (MACS&more, Vol.8, 2/2004).
In case the starting material is highly limited or the researcher is interested in the transcriptome of only a few or even a single cell, the SuperAmp™ Service is available (see below). |
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| Can you help us establishing PIQOR Microarray technology in our lab? |
| Yes, Miltenyi Biotec offers in-depth training courses where you will learn the practical processing of microarrays in the lab as well as the theory of PIQOR™ Microarray analysis. For more information please contact us (http://www.miltenyibiotec.com/nn.645). |
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| Do I need special equipment for the readout of PIQOR Microarrays? |
| No, the PIQOR™ Microarrays are produced on standard microscope glass slides that can be inserted in microarray scanners offered by, e.g. Agilent, GE-Healthcare, or Applied Biosystems. |
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| What kind of gene expression profiling service is offered? |
The highly flexible, cost-effective gene expression profiling service is tailored to the specific needs of the researcher. Simply send your samples, for example tissue, cells, or blood, and receive ready-to-publish results: an extensive written report including all experimental steps and performed data analyses. All relevant data files are provided on CD-Rom.
We perform the experiments, starting from RNA extraction and labeling to hybridization, scanning, and bioinformatics data analysis.
Microarray Service includes:
- Consulting: microarray selection and experimental design
- RNA extraction, quality control (Agilent 2100 Bioanalyzer, Agilent Technologies)
- Optional: Amplification; SuperAmp™ Service
- Labeling, quality control
- Hybridization of topic-defined or custom PIQOR or Agilent Whole Genome Microarrays
- Readout and primary data analysis
- Documentation, written report, all relevant data files on CD-Rom
- Optional: Advanced bioinformatics analysis including cluster- and pathway analysis for large-scale projects |
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| What exactly are the Bioinformatic Services offered by Miltenyi Biotec? |
The available Bioinformatics Services are:
- Cluster Analysis
- Discriminatory Genes Analysis
- Pathway Analysis
Cluster Analysis—hierarchical grouping of relevant genes
Cluster analysis groups all relevant genes according to the similarity of their expression profiles. Thus, the genes are ordered in a hierarchical tree, where similarly activated genes are located next to each other, whereas differently expressed genes are placed distant from each other. Noticeable groups of genes for a common biological context can be examined.
Discriminatory Genes Analysis—"Fingerprint" for a specific disease
Discriminatory genes are genes that behave differently under the conditions of interest. For example, all discriminatory genes that are induced under test condition A but are repressed under control condition B.
Pathway analysis—coregulated gene groups
Based on cluster or discriminatory genes analysis, the biological annotation analysis places coregulated genes in groups according to similar biological functions or characteristics. The proprietary TreeRanker Software identifies significantly different gene groups if they are involved in same pathways, share common protein motifs, correlate with same disease or cellular process, etc.
In a next step, the Signaling network analysis visualizes if and how the genes of interest—or their protein products—could interact. Common direct regulators or downstream targets can be detected, and activation or inhibition is distinguished. This analysis is based on biomedical literature using the PathwayArchitect™ Software, Stratagene, for pathway analysis and visualization.
The bioinformatics specialists update their proprietary as well as commercially and publically available data sources constantly. |
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| What is the required processing time for the Microarray Service? |
The time depends on the individual research project, especially if new, customized microarrays or gene fragments have to be produced:
- Up to 20 PIQOR™ or Agilent Microarrays are processed in about 2–3 weeks. This includes RNA extraction, quality controls, labeling reaction, hybdridization of microarrays, readout of scans, extraction of primary data as well as the generation of normalized ratios and written report.
- Custom PIQOR™ Microarray experiments demand approx. 4–6 weeks for the production of the microarrays if the gene fragments are selected from the established PIQOR™ Gene Collection.
- Additional gene fragments require extra time for the generation process, this would take about 6 –8 weeks in total. The average success rate is 80–90%. |
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| What are the Agilent 4x44K One-Color and Two-Color Whole Genome Microarray experiments? |
Miltenyi Biotec is a certified Agilent Services Provider and offers high-quality gene expression analysis services based on Agilent whole-genome microarrays.
The Agilent Multi-Pack Microarrays can be used for both one- and two-color experiments depending on the specific applications and needs of the researcher.
For one-color microarray analysis, the individual sample is labeled and then hybridized on one microarray. This simple and flexible experimental setup compares the measured gene expression of a microarray directly across other microarrays. Data constistency improves by performing sufficient technical and biological replicate assays.
In a two-color procedure, two samples are individually labeled with different fluorochromes, e.g. Cyanine-3 (Cy3) and Cyanine-5 (Cy5), and then hybridized on the same microarray. This experimental design allows a direct comparison between control versus experimental sample, thus minimizing variability between slides.
Features of the 4x44K Multi-Pack format
• High sensitivity due to small microarray footprint
• More arrays per slide reduces the cost per experiment.
• An improved dynamic range of 5.0–6.0 orders of magnitude provides wide range of detection.
• Agilent Spike-in controls and quality control reporting for high confidence in results
Agilent Whole Genome Microarrays for human, mouse, and rat
Agilent 60-mer oligonucleotide microarrays are printed with SurePrint Technology. The whole-genome microarrays cover more than 41,000 genes and transcripts. The representative sequences are derived from leading public databases including RefSeq, Ensembl, Unigene, UCSC Goldenpath, and others. Over 70% of the 60-mer probes have been verified and optimized by Agilent‘s empirical validation process.
In addition to human, mouse and rat, further microarray formats for various species can be processed within the custom Agilent Service such as rhesus monkey, dog (Canis familiaris), Xenopus laevis, zebrafish (Danio rerio), etc. |
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| How complex is the amplification of RNA from a limited amount of material? |
| The user manual of the PIQOR™ Microarray Kit contains detailed, hands-on protocols, with an emphasis on how to amplify low amounts of RNA. Please note the SuperAmp Service below. |
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| What is the SuperAmp™ Service? |
The SuperAmpTM Service is an RNA amplification service for customers with 1–100,000 cells as starting material, for example, cells sorted with MACS® Technology, laser capture microdissected cells, cells sorted by flow cytometry, tissue biopsies, and primary cells can be analyzed using the SuperAmp Service.
Note: SuperAmp Service is only available in combination with the PIQOR Microarray Service Plus Amplification or the Agilent Whole Genome Microarray Service. |
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| What is PrepProtect™ Stabilization Buffer? |
One main difference of RNA to DNA is the hydroxyl group at the 2’ position in the sugar pentose ring - and not just an hydrogen as in the DNA. With the second hydroxyl group, RNA is less stable and more prone to chemical and enzymatical degradation.
PrepProtect is a non-toxic reagent for stabilization of RNA in biological cell and tissue samples. Just add PrepProtect to the sample: The solution penetrates cells or tissues and it inhibits RNases. The RNA integrity is maintained, preserving the gene expression profile if immediate cell lysis is not possible. This stabilization buffer even seeps through previously frozen material and protects RNA during thawing. Even DNA and denatured proteins are preserved. Also, tissue structure is softened, and its lysis is facilitated.
High-quality RNA is a prerequisite for many gene expression profiling and cDNA cloning
techniques like Microarray analysis, real-time PCR, RNase protection assays, Northern blot analysis, and cDNA library construction.
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
MACS is a registered trademark of Miltenyi Biotec GmbH. PIQOR, SuperAmp, and PrepProtect are trademarks of Miltenyi Biotec GmbH.
PathwayArchitect is a trademark of Stratagene. |
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