Service

Sample quality control - Bioanalyzer

A key point in microarray analysis is the quality of the sample used. The Agilent 2100 Bioanalyzer microcapillary chip gel electrophoresis system is suitable for the measurement of the purity and composition of the isolated nucleic acids, which is the basis for ensuring the quality of microarray analysis. The procedure is equivalent to gel electrophoresis, however it is two fold more sensitive. Furthermore, due to the automatic data collection, processing and storage it is a sensitive and reliable method. The device, operating on the basis of microcapillaries, is suitable for receiving 12/11 samples simultaneously. Using only 1ul of sample, the measurement takes only 30 minutes (referred to 12 or 11 samples). Detection of the individual fragments is achieved by the measurement of the intercalated fluorescent dye by a laser beam.

Naturally, the method can be used not only for microarray studies. Any case, the quality of the samples should be checked when one wants to know if the sample is intact or not (e.g. biobank, qPCR, WGA, etc.).

For the purification of nucleic acid with good quality (mainly RNA) is essential to choose the appropriate isolation method.

We undertake total RNA, miRNA and genomic DNA isolation of a wide variety of source material, even from small amounts of sample or samples that require specific conditions (e.g. fibrous tissue, high-fat tissue, etc.).

Expression microarray

One of the greatest innovations of the past decades is the development of expression microarrays. As a result, previously unimaginable amount of data became available and novel physiological and pathological processes could be identified by their analysis.

Chromoscience Ltd provides complete gene expression services from the design of the experiment and sample isolation to carrying out the measurement and complex data analysis. Measurements are performed with the Agilent Technologies System, one of the world’s most cutting-edge microarray system manufacturer, thus insuring very precise results. Gene expression microarray is a hybridization-based system suitable for parallel analysis of multiple sequences, which enables the simultaneous investigation of the expression of thousands of genes. The oligonucleotide probes specific to the genes of interest are fixed on a solid support (glass slide), then the fluorescently labelled cRNA prepared from the isolated mRNA is hybridized to the slide and tissue specific expression of genes can be assayed. Microarray assays provide quantitative results on gene expression patterns.

Some of the numerous advantages of Agilent gene expression platform are highlighted below:

  • 60 mer length oligonucleotide probes providing high specificity
  • High dynamic measuring range (> 5 logs)
  • Very high accuracy, high degree of correlation with Taqman RT-PCR (r>0,9)
  • Reproducibility (covariance of signal values <10%)
  • Fully customized content in multiple formats
  • Even a small amount of starting material is sufficient (50ng of total RNA)

On the one hand the " ink jet " print technology provides great flexibility while on the other hand allows not only the completion of commercially available microarray measurements, but also targeted expression measurements focusing to gene groups, designed according to individual needs. In situ oligo synthesis based on SurePrint technology enables rapid change of microarray composition, thus keep up with continuous growing of genetic content and gene annotation. Furthermore it ensures a unique flexibility for the production of custom arrays. A website with specific data security is available (Agilent earray) for the design of custom arrays, where the user can design and has chip manufactured with quality conditions mentioned above.

Features of our most commonly used expression microarray formats:

  • human 4x44K
    • The 4x44K array allows the measurement of 4 samples on a slide
    • 60 mer length probes printed with Sure Print technology provide a high degree of specificity
    • It offers the possibility of detecting 27958 targets based on Entrez Gene database
    • The content is edited based on RefSeq, Ensembl, UniGene, and GenBank databases covering the entire human transcriptome
    • or expression measurement always the latest Agilent array format is used
  • mouse 4x44K
    • This array format enables the measurement of 4 samples on a slide
    • 60 mer length probes printed with Sure Print technology provide a high degree of specificity
    • Possibility of 39430 target detection based on Entrez Gene database
    • The content is edited based on RefSeq, Ensembl, RIKEN, UniGene, and GenBank databases covering the entire mouse transcriptome
    • For expression measurement always the latest Agilent array format is used

Expression measurements of different model organisms, such as Arabidopsis, Caenorhabditis, Drosophila, Magnaporthe, or Xenopus are available in the context of service options. Please contact us and inquire about expression measurement opportunities associated with other model organisms.

Agilent One- or Two-Color Options for Targeted Analysis:

Agilent's Dual-Mode Gene Expression Analysis Platform gives researchers the ease of one-color experimental design and the resolving power of two-color detection in a single platform. In a direct comparison of two RNA samples, two-color microarrays usually give the most accurate results, particularly for small changes of expression levels, because two RNAs are co-hybridized to the arrays. However, for experiments involving multiple group comparison of large number of samples, experimental design for one-color microarrays is more straightforward and cost-efficient. Data generated by both approaches provide excellent platform performance and superb quality control.

Azonban olyan vizsgálatokban, ahol több kísérleti csoport van, az egyszínű opció a legegyszerűbben magyarázható és legköltséghatékonyabb választás. Mindkét opció kiváló rendszerteljesítményt és felsőfokú minőséget nyújt.

miRNA microarray

The evolutionarily highly conserved miRNAs are ubiquitous regulatory molecules which do not encode proteins, however, due to their interaction with mRNA play a key role in the posttranscriptional regulation of gene expression. Increasing data support their pivotal role in basic biological functions e.g. mitosis, differentiation, apoptosis, and lipid metabolism.

Chromoscience Ltd. offers the precise and reliable Agilent miRNA expression system for customers, which provides the following options:

  • Detection of miRNAs in the sample
  • Starting from total RNA which eliminates distortions/inaccuracy during miRNA isolation
  • Content is edited based on Sanger miRBase database
  • Optimum sensitivity and selectivity provided by sophisticated probe design and unique marking
  • Widest dynamic measuring range (> 5 log) ensures the detection of low and high- expressed molecules in order to achieve the most relevant biological data
  • Fully customizable content in multiple formats for the most flexible experiment design
  • 100 ng of total RNA is sufficient
  • Available in human, mouse and rat species
  • Multiplex-format: 8x15K

Features of our most commonly used miRNA microarray formats:

  • human 8x15K
    • The 8x15K array enables the measurement of 8 samples on a slide
    • 60 mer length probes printed with Sure Print technology provide a high degree of specificity
    • 866 human and 89 human viral miRNAs can be measured simultaneously
    • Content is edited based on Sanger miRBase database
    • For expression measurement always the latest Agilent array format is used
  • mouse 8x15K
    • This array format allows the measurement of 8 samples on a slide
    • 60 mer length probes printed with Sure Print technology provide a high degree of specificity
    • 627 mouse miRNAs and 39 mouse virus miRNAs can be measured simultaneously Content is edited based on Sanger miRBase database
    • For expression measurement always the latest Agilent array format is used

ChIP-on-chip

By the help of ChIP-on-chip method which combines chromatin immunoprecipitation ("ChIP") and microarray ("chip") technology in vivo DNA-protein interactions can be examined. This method is suitable for example, monitoring the localization and regulation of DNA-binded transcription factors, as well as testing DNA repair and replication, histone modification or DNA methylation.

Features of our most commonly used Agilent promoter microarrays:

  • They contain optimized probes validated to ChIP-on-chip technology, which are designed specifically for the determination of DNA-binding protein location
  • Content was edited based on UCSC human genome and UCSC Mus musculus databases
  • In addition to the most common human and mouse microarray formats it is available in other species (yeast, nematodes, Drosophila, zebrafish, Schizosaccharomyces pombe)
  • The human ENCODE ChIP-on-chip arrays are specifically developed for the identification of DNA-binding proteins in coding regions

In case of ChIP-on-chip measurements our services cover the DNA hybridization and marking steps.

aCGH

Array CGH is one of the most modern methods for detection of chromosome copy number and/or structural changes (translocations, deletions, microdeletions, amplifications). This microarray-based technology enables the completion of a large number of studies at the same time in an integrated system, which ultimately provides more accurate information than cytogenetic tests about the possible chromosomal changes.

DNA used in the tests can be derived directly from blood, amniotic fluid, chorionic villus, bone marrow, and tissue sample. The method is fast, and does not involve the use of cell cultures, thus one may obtain perfect results even in cases when classic cytogenetic methods cannot be clearly evaluated. In the experiment test (e.g. from tumor tissue) DNA is marked with a green fluorescent dye, while the intact (control) DNA derived from tissue indicated with another red fluorescent dye and are hybridized to a glass slide to which a large number of cDNA or genomic DNA with known sequence are fixed (DNA chip, DNA microarray). After reading by a laser scanner differences in color can be evaluated using a computer.

In the context of our services we offer Agilent’s different resolution whole genome aCGHs developed for research purposes or designed according to the customer's needs for human, mouse and rat species.

In some cases, for testing certain biological samples we recommend international standard cytogenetic arrays (ISCA, International Standard Cytogenetic Array) intended with clinical focus.

CytoChip ISCA arrays are designed to investigate constitutional disorders through a combination of increased probe density in regions or genes associated with known constitutional disorders.

When the quantity and/or quality of the test sample does not allow the execution of conventional cytogenetic tests (FISH and G-banded karyotyping) and aCGH measurements, the Cytochip Focus array family is the ideal choice for analysis. The Cytochip Focus is a well-tried ultra-sensitive BAC array, which enables the detection of bigger chromosome structural changes even in case of clonal cell populations. It serves with reliable results even from 50 ng of DNA of a weak quality, eliminating cell culturing and whole genome amplification. Thus, any artificial chromosome errors created by cell culture procedure can be avoided. This occur frequently in hematologic samples, thereby obscuring the real biological state.

The CytoChip Cancer array design is highly enriched in disease regions and is exon-focused. Each CytoChip Cancer design includes more than 20,000 disease-targeted oligo probes, covering 670 cancer genes, including those which are typically amplified, deleted and translocated in cancers.

qPCR

Quantitative Polymerase Chain Reaction

 

The most complicated part of gene expression and miRNA microarray studies is perhaps the processing and interpretation of results and validation of the data obtained. Based on the customer's needs we provide verification of experimental data (mRNA, miRNA) by Quantitative Polymerase Chain Reaction (qPCR).

The method is based on the general PCR, its main characteristic however, that the quantitative analysis of amplified DNA/cDNA happens „real-time” while the reaction occurs. Two procedures are used for detecting the product:

  • application of non-specific fluorescent dyes: these are able to intercalate into any double-stranded DNA molecule
  • using sequence-specific DNA probes: these are built up from oligonucleotides labeled by fluorescent reporter, which signal becomes detectable only after hybridization of the probe to its complementary sequence

Both methods are available in our laboratory and in case of need we can provide assistance in selecting and designing sequence-specific primers used in the reaction.