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Gene Core – Quantitative and digital PCR

About us

The qPCR and dPCR Core Facility is one of Europe’s leading academic service providers specialized in high-throughput gene expression analysis using real-time quantitative PCR (qPCR). We have broad experience in quality control (QC e.g. Fragment Analyser) in a single cell analysis (automated cell picking ALS Cellcelector), high-throughput and digital PCR (Fluidigm Biomark, BioRad QX200 Droplet Digital PCR System) and NGS library preparation.

Potential for cooperation

We offer collaboration both to internal and external researchers from the Czech Republic and we are also open to international cooperation. Our aim is to make state-of-the-art qPCR and dPCR technologies and know-how related to nucleic acids analysis available to BIOCEV and academic researchers under attractive conditions. We can contribute to clients’ workflow through the extraction of samples, control of nucleic acid quality, reverse transcription to qPCR or dPCR, data analysis and any downstream applications. We also offer academic researchers the possibility to perform their own experiments at our facility under our supervision.

Please see also the OFFICIAL WEBSITE 


Team and contacts

Místnost
G1.047, G1.046
  • Mgr. David Švec, Ph.D.

  • Postdoc of Laboratory of Gene Expression, consultant for single-cell analysis
  • +420 325 873 748
Místnost
G1.048

Where to find us in BIOCEV center

Mapa1NP-PCR


Equipment


  • The AVISO CellCelector (Automated Lab Solutions)

    Aviso_CellColector Advalytix3 fbcb50ccc1

    The AVISO CellCelector is a freely configurable tool for the automated transfer of single cells and cell colonies. The patented harvest process allows a gentle cell uptake directly from the culture plate without pre-treatment. Using this technology, highest survival rate and cell integrity are guaranteed.  This service is only with CF operator. Please clame the service 48 hours in advance.

     

    Additional information

    Video

  • 2100 Bioanalyzer (Agilent)

    The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells on a single platform. Results are delivered within 30-40 minutes in automated, high quality digital data. You can analyze 11-12 samples in one run. Report with RIN numbers is a part of the analysis.

    Consumables for RNA or DNA are available at CF.

    The array for RNA std analysis of 12 samples, which is NOT included in the price: 906 CZK without VAT.

    If you would like to run other kind of analysis, contact Vlasta Korenková.

    Additional information

  • LightCycler 480 System (Roche)

    The LightCycler 480 Real-Time PCR System is a multiwell-plate based real-time PCR platform which is used for highly accurate qualitative and quantitative detection of nucleic acids and genotyping. 96 and 384 well plates are available at CF to purchase as well as mastermixes for RT-qPCR. The instrument is available after training for self use.You can combine this service with automatic pipetting of 384-well plate using Epmotion P5073 (Eppendorf).

     

    Properties of LC 480:

    Light source: Xenon lamp
    Detection: CCD camera
    Excitation filters: 440 nm, 465 nm, 498 nm, 533 nm, 618 nm
    Emission filters: 488 nm, 510 nm, 580 nm, 610 nm, 640 nm, 660 nm
    Silver thermal block
    LightCycler 480 Software provides versatile solutions for the most common real-time applications
    High Resolution Melting Analysis available

     

    You can purchase in CF:

    1x 384 PCR plate with foil: 221 without VAT
    1x 96 PCR plate with foil: 120 without VAT

    ADDITIONAL INFORMATION: https://lifescience.roche.com/shop/products/lightcycler14301-480-instrument-ii

    VIDEO: www.youtube.com/watch?v=FwPzrFN9eV4

  • QX200 droplet digital PCR system (Bio-Rad)

    The ddPCR System provides absolute quantification of target DNA or RNA molecules for EvaGreen or probe-based digital PCR applications without a need for a standard curve.

    All material for ddPCR experiment is available at CF.

    It is necessary to book C1000 for 2 hours before ddPCR.

    We offer quantification with EvaGreen, probes and NGS libraries.

    There are different costs for different amount of samples: 8,16, 24, 48, 96.

    ADDITIONAL INFORMATION: www.bio-rad.com/en-us/product/qx200-droplet-digital-pcr-system?WT.srch=1&WT.mc_id=aw-dbc-EM-digital_pcr_brand_gold&WT.knsh_id=02adc4da-a9f3-4021-ae56-9d815896d1f4

    VIDEO: www.youtube.com/watch?v=Qwma-1Ek-Y4

  • Qubit® 2.0 Fluorometer (life Technologies)

    The Qubit 2.0 Fluorometer quantitates DNA, RNA, and protein with unprecedented accuracy, sensitivity, and simplicity. Qubit® fluorometric quantitation uses Qubit® assays that contain advanced dyes that only fluoresce when bound to DNA, RNA, or protein. This specificity allows you to get very accurate results because Qubit technology only reports the concentration of the molecule of interest, not contaminants.

    Asssays for measurements of DNA, RNA and miicroRNA are available at CF: 1 reaction = 13 CZK.

    ADDITIONAL INFORMATION: www.thermofisher.com/cz/en/home/life-science/laboratory-instruments/fluorometers/qubit/qubit-technical-resources/previous-qubit-models.html

    VIDEO: www.youtube.com/watch?v=RRKZN–7jqg

  • QIAcube (Qiagen)

    QIAcube processes QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow. No change of purification chemistry is required, assuring fast startup and immediate results. All steps in the purification procedure are fully automated – and up to 12 samples can be processed per run. Tips for the instrument are available.

    Kit for isolation is not included.

    ADDITIONAL INFORMATION: www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/qiacube/

    VIDEO: www.qiagen.com/media/product-tools/flash/qiacube/360/index.html

    https://youtu.be/Li4lSMqSNO8

  • GentleMACS™ Dissociator (MACS Miltenyi Biotec)

    The gentleMACS™ Dissociator is a benchtop instrument for the automated dissociation of tissues. Two types of unique gentleMACS Tubes used with the instrument enable the time-saving and easy dissociation or homogenization of tissues in a closed system. C Tubes are used for the gentle preparation of single-cell suspensions from tissues, whereas M Tubes are used for the homogenization of tissues or cells. A single sample or two samples in parallel can be processed.

     

    Consumables are available at CF:

    C tubes for dissociation of tissues to single cells. NO FEE for test runs.

    M tubes for the complete dissociation of tissues. NO FEE for test runs.

    ADDITIONAL INFORMATION: www.miltenyibiotec.com/en/products-and-services/macs-sample-preparation/sample-dissociation/gentlemacs-dissociators/gentlemacs-dissociator.aspx

    VIDEO: www.youtube.com/watch?v=vfAdmYCiaQE

  • Gel Doc EZ system (Bio-Rad)

    The Gel Doc EZ system (Bio-Rad) is a compact and automated gel imaging instrument designed to yield publication-quality images and analyzed results with just the push of a button. The Gel Doc EZ system provides unparalleled flexibility with the use of four application-specific trays:

    - UV tray for ethidium bromide staining of DNA gels and fluorescence imaging

    - White tray for Coomassie, copper, silver, and zinc stains

    - Blue tray for nucleic acid applications with SYBR® stains (not available now)

    - Stain-free tray for stain-free imaging with Bio-Rad stain-free gels

    ADDITIONAL INFORMATION: http://www.bio-rad.com/en-cz/product/gel-doc-ez-system?WT.knsh_id=02adc4da-a9f3-4021-ae56-9d815896d1f4&WT.mc_id=aw-pqd-EM-geldocez_brand_gold&WT.srch=1

    VIDEO: https://www.youtube.com/watch?v=JiV51QfrvPU

    COST: FREE MEASUREMENTS

  • Fragment Analyzer (Advanced Analytical Technologies)

    Fragment Analyzer (Advanced Analytical) will automate capillary electrophoresis. Fragment Analyzer is used and recommended by Illumina™ as an automated system for the quantification and qualification of NGS libraries, gDNA and RNA.

     

    -Run 12- or 96-capillary plates

    -Resolves fragments from 10 bp to 40,000 bp

    -Get resolution down to 2 bp for fragments

    -Detection starts at 5 pg/μL

    Various kits are available at qPCR and dPCR CF.

    ADDITIONAL INFORMATION: http://aati-us.com/product/fragment-analyzer

    VIDEO: http://aati-us.com/product/fragment-analyzer

  • Experion Automated Electrophoresis Station (Bio-Rad)

    The Experion automated electrophoresis station performs all of the steps of gel-based electrophoresis in one compact, durable unit.It automates analysis by combining electrophoresis, staining, destaining, band detection, and imaging into a single, 30 minute step.You can analyze 11-12 samples in one run. Report with RQI numbers is a part of the analysis.

     

    Consumables for RNA or DNA are available at CF. The run time for one analysis is 30 minut (make 30 min. booking)

    The array for RNA std analysis of 12 sample, which is NOT included in the price: 906 CZK without VAT.

    If you would like to run other kind of analysis, contact Vlasta Korenková.

    ADITIONAL INFORMATION: www.bio-rad.com/en-cz/category/experion-automated-electrophoresis-system

    VIDEO: www.youtube.com/watch?v=2VCAZFkhxO8

  • CFX96 Real Time PCR Instrument (Bio-Rad)

    A cycler for quantitative real-time PCR from Bio-Rad. You can obtain low-profile 96 well plates at core facility. The instrument is available for researches after training.

    Properties of CFX 96:

    Light source: 6 LEDs in optics shuttle
    Detection: 6 photodiodes
    Excitation wavelenghts: 450–684 nm
    Emission wavelenghts: 515–730 nm
    Gradient range: 30–100°C, Maximum gradient span 24°C
    Software for gene expression analysis
    Precision melt software: Enables High Resolution Melt analysis, min. step for melt curve 0.2°C

    Consumables that are not included in price can be bought at CF:

    1x 96 PCR plate + 1x foil: 174 CZK without VAT
    1x strip (for 8 samples) + 1x cap: 33 CZK without VAT

    ADDITIONAL INFORMATION: www.bio-rad.com/en-us/product/cfx96-touch-real-time-pcr-detection-system

    VIDEO: www.youtube.com/watch?v=NY9mj-qILDU

  • epMotion P5073 Automated Pipetting System (Eppendorf)

    The Eppendorf epMotion 5073 workstation enables fast and reproducible liquid handling by way of pipetting, dispensing, and other dispensing variants. It enables quick filling of plates in the 384-well scale. It is also possible to fill an MTP 384-well from other formats (eg, 4 x MTP 96-well).

    Special pipette tips are available at CF and calculated in the price. It is possible to buy mastermixes at CF. 1 tip = 1,10 CZK

    ADDITIONAL INFORMATION: https://online-shop.eppendorf.cz/CZ-en/Automated-Pipetting-44509/Liquid-Handling-Workstations-44510/epM5073-PF-20687.html

    VIDEO: www.youtube.com/watch?v=REVXumkaYrg

    www.youtube.com/watch?v=vgJdySaNPXY

  • C1000 Thermal Cycler (Bio-Rad)

    C1000 is a thermal cycler that offers a gradient 96-well fast module. It supports deep-well PCR plates.
    Gradient temperature range 30-100°C
    Gradient temperature differential 1-24°
    Gradient accuracy ±0.2°C of programmed temperature at end rows

    PCR plates and sealing foils are available at CF:
    1 PCR plate with foil —– 172 CZK without VAT
    1 strip with 1 cap —– 32 CZK without VAT
    You can obtain mastermix as well the price on request

  • Nanodrop 8000 (Thermo Scientific)

    Thermo Scientific NanoDrop 8000 UV-Vis Spectrophotometers are used  to measure between 1 and 8 microvolume nucleic acid and protein samples simultaneously.  The patented sample retention system allows you to assess the concentration and purity of samples as small as 1µL.

    Properties:

    • Choose to measure form 1 to 8 samples in one measurement cycle
    • Wide spectral range (220 – 750nm) for measuring a variety of samples types
    • Patented sample retention system automatically optimizes pathlength to accommodate low and high concentrations (2.5 to 3,700 ng/µL dsDNA), no dilution is required
    • Pre-configured methods for DNA, Protein A280, Microarray, Protein and Labels, Pierce 660, Bradford, BCA, and Lowry

     

    You can measure for free if you bring your our pipette tips. Please do not bring PCR products or plasmids. These can be measured at G1.049.BTU. If you would like to connect your printer to the instrument, please contact Vlasta Korenkova.

    ADDITIONAL INFORMATION: www.nanodrop.com/productnd8000overview.aspx

    VIDEO: www.youtube.com/watch?v=WfirigiekwM

  • BioMark qPCR System (Fluidigm)

    High-throughput qPCR System BioMark (Fluidigm) uses integrated fluidic circuits known as dynamic arrays and digital arrays. These innovative products allow scientists to practice tried-and-true techniques, such as TaqMan assays or EvaGreen chemistry, while realizing a previously unachievable throughput. Researchers save on reagents, pipette tips, time, and on the upkeep of expensive liquid-handling robots. 2 ul of the sample are sufficient for the analysis.

    Available formates of chips:

    Flex Six™ IFC for Gene Expression: 6 x reusable chip that combines 12 samples with 12 assays (6x 144 reactions)
    96.96 Dynamic Array IFC for Gene Expression: combines 96 samples with 96 assays (9216 reactions)
    96.96 Dynamic Array IFC for Genotyping: combines 96 samples with 96 assays (9216 reactions)
    48.48 Dynamic Array IFC for Gene Expression: combines 48 samples with 48 assays (2304 reactions)
    48.48 Dynamic Array IFC for Genotyping: combines 48 samples with 48 assays (2304 reactions)
    12.765 Digital Array IFC: for dPCR of 12 samples
    48.770 Digital Array IFC: for dPCR of 48 samples

    Instrument characteristics:

    Light source Xenon lamp with excitation filters
    Detection CCD camera
    Excitation wavelenghts 465-505 nm, 510-550 nm
    Emission wavelenghts 500-550 nm, 540-600 nm
    Examples of supported probe types FAM-MGB, VIC-MGB, FAM-TAMRA, FAM-non fluorescent quencher

    Applications:

    Gene expression Microarray validation, Single cell gene expression, Drug efficacy and safety
    Digital PCR Absolute quantitation, Mutation detection
    Genotyping SNP association, Single cell

    Arrays are available at CF.

    Additional information

    Video 1

    Video 2

    Video 3

  • TissueLyser LT (Qiagen)

    For low- to medium-throughput sample disruption of tissues for molecular analysis.

    Simultaneous disruption of up to 12 samples in 2 ml tubes

    Coolable adapter to prevent biomolecule degradation

    Before use, please ask for information about beeds.

    You can get stain steal beads in diametter 5 mm (Cat No./ID   69989) in CF. One bead per 23 CZK.

    During reservation, indicate a time of use, minimum is 15 min.

    ADDITIONAL INFORMATION: www.qiagen.com/us/shop/automated-solutions/sample-disruption/tissuelyser-lt

    VIDEO: www.qiagen.com/media/product-tools/flash/TissueLyserLT/360/index.html

    https://www.youtube.com/watch?v=31O6AKamfks

Our Services

Biostatistical service and experimental design
Isolation of single cells
Isolation of nucleic acids
Integrity and quantity control of nucleic acids

  • Quantity control of nucleic acids with a spectrophotometer Nanodrop 8000 (Thermo Scientific)
  • Quantity control of nucleic acids with a fluorospectrophotometer Quibit 2.0 (Life Technologies)
  • Integrity control of nucleic acids with an Experion Automated Electrophoresis Station (Bio-Rad)
  • Integrity control of nucleic acids with a Bioanalyzer 2100 (Agilent)
Reverse transcription
Quantitative real-time PCR

  • Absolute quantification
  • Relative quantification
  • Selection of appropriate reference genes for your system
  • Control of contamination with genomic DNA
  • Validation of new primers and probes according to MIQE
  • Control of inhibition in RT or in qPCR
High-throughput qPCR: BioMark (Fluidigm)

  • Gene expression: 9216 qPCR reactions with GE 96.96 or 2304 qPCR reactions with GE 48.48
  • Genotyping: 9216 qPCR reactions with GT 96.96 or 2304 qPCR reactions with GT 48.48
  • Preamplification
Digital PCR

  • Droplet digital PCR with QX200 (Bio-Rad) for 8 – 96 samples
  • Microfluidic digital PCR with BioMark (Fluidigm) for 12 or 48 samples
Special aplication: Single cell qPCR analysis
Automatization

  • Automatic isolation of nucleic acids with QiaCube (Qiagen) for 12 samples
  • Automatic pipetting and preparation of dilution series with epMotion P5073 (Eppendorf)

 

 

Biostatistical service and experimental design

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Experimental design is the process of planning a study to meet specified objectives.  Planning an experiment properly is very important in order to ensure that the right type of data and a sufficient sample size and power are available to answer the research questions of interest as clearly and efficiently as possible.

Our biostatistician will help you to design your experiment properly, advice what kind of controls to use, how to collect samples correctly, calculates an optimal sample size and suggests the best approach. The biostatistician will also manage large-scale biological datasets and analyze them.

For qPCR data preprocessing and analysis we use software GenEx Enterprise (MultiD Analyses) and SAS.

REQUEST A MEETING (Wednesday from 13.00 to 16.00, each session ½ hour)

ORDER a Genex software from us

 

Isolation of single cells

  1. We offer using the AVISO CellCelector with our experienced laboratory staff. It is a freely configurable tool for the automated transfer of single cells and cell colonies. The labeled cells are picked individually according to the characteristics you supply. We can pick either attached cells from petri dishes, from a slide, from a filter or cells from a suspension.ADDITIONAL RESOURCES: http://www.als-jena.com/fileadmin/user_upload/bilder/PDFs/Startseite/Mesenchymal_single_cell_Application_Note.pdf1
  2. We offer a single cell isolation from tissues with a gentleMACS™ Dissociator. The instrument offers optimized gentleMACS Programs for a variety of specific applications. The standardized tissue dissociation or homogenization procedures make for reliable and reproducible results. Researchers can use instrument after training or we can isolate your cells for you in form of a service. Tubes C and M are available at CF, come to test it for FREE.ADDITIONAL RESOURCES: https://www.miltenyibiotec.com/~/media/Files/Navigation/Research/Cardio/gentleMACS_brochure.ashx2

Isolation of nucleic acids

Come to disscuss the posibility how to isolate your sample.

We provide reproducible isolation of DNA, RNA, microRNA or cfDNA with Qiagene isolation kits using automatic isolation system QiaCube.

As a part of the service, we will measure the quantity and quality of your NA.

We can also provide the measurement integrity of your RNA

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Integrity and quantity control of nucleic acids

Quantity control of nucleic acids with a fluorospectrophotometer Nanodrop 8000 (Thermo Scientific)

As a part of the isolation service, we will measure the quantity and quality of you NA with Nanodrop 8000 spectrophotometer, which can realiable concentrations from 2.5 to 3,700 ng/µL dsDNA.

A measured values and their optimal ranges:

  • DNA a RNA (260nm) > 10 ng/ul
  • A good quality DNA sample should have a A260/A280 ratio of 1.7-2.0 and an A260/A230 ratio of greater than 1.5
  • A good quality RNA sample should have a A260/A280 ratio of 1.8-2.0 and an A260/A230 ratio of 1.8 – 2.245

ADDITIONAL RESOURCES:

How to interpret RNA peaks: http://www.u.arizona.edu/~gwatts/azcc/InterpretingSpec.pdf

What contamination of your NA looks like: http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf

Importance of 260/230 and 260/280 ratios: http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf

Troubleshooting for RNA isolation: http://bitesizebio.com/2345/troubleshooting-rna-isolation/

 

Quantity control of nucleic acids with a fluorospektorphotometer Quibit 2.0 (Life Technologies)

We offer quantification of nucleic acids using The Qubit® Fluorometer that utilizes fluorescent dyes that are specific to the target of interest. Qubit® assay kits fluoresce only when bound to the selected molecule—DNA, RNA, or protein—in your sample, even at low concentrations.  It relies on reading two standards for calibration every time that you make measurements. From 1 to 20 µl of DNA sample is required, with 2 µl being the amount typically used by the lab. Researchers can use instrument after training or we can isolate your cells for you in form of a service.

Advantages:

  • Ideal for measurement of low concentrations NA below 5 ng/ul because UV spectrophotometry often does not have the sensitivity to accurately measure low concentrations of DNA and RNA.
  • The effective range covers a sample concentration range of  10 pg/μL to 1 μg/μL DNA.
  • Qubit® RNA assays are designed to be specific and accurate for RNA even in the presence of a 1:1 mixture of RNA and DNA.
  • The Qubit® Fluorometer supports assays for high-sensitivity or broad-range dsDNA and RNA quantitation. Assays for oligos, ssDNA, protein, and microRNA quantification are also available.2

 

ADDITIONAL RESOURCES: https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/cell-tissue-analysis/qubit-all-file-types.par.0519.file.dat/qubit-2-fluorometer-user-manual.pdf

https://www.thermofisher.com/cz/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-assays.html

https://www.youtube.com/watch?v=RRKZN–7jqg

COST OF A SERVICE: 13 CZK without VAT per a sample

 

Integrity control of nucleic acids with an Experion Automated Electrophoresis Station (Bio-Rad)

We offer integrity control of nucleic acids using Experion Automated Electrophoresis Station that replaces regular gel analysis and offers higher sensitivity. The instrument combines quantitation and quality assessment in a single step. From 1.5 to 2.5 µl of NA sample is required. Kit for measuring RNA is available at CF. 12 samples of RNA or 11 samples of DNA can be measured in one chip, respectively. Researchers can use instrument after training or we can measure integrity your samples for you in form of a service.

Integrity of RNA: The RQI (RNA Quality Indicator) method returns a number between 10 (intact RNA) and 1 (highly degraded RNA) for each eukaryotic RNA sample run on an Experion RNA StdSens or HighSens analysis chip. The RQI measures RNA integrity by comparing the electropherogram of RNA samples to a series of standardized degraded RNA samples.

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DNA analysis: The Experion DNA 1K and DNA 12K analysis kits allow analysis of DNA samples with size ranges of 15–1,500 bp and 50–12,000 bp, respectively. These DNA assays provide high sensitivity and excellent resolution (down to 5 bp) over a broad dynamic range. Consuming only 1 µl of sample for each analysis, the Experion automated system can analyze 1–11 samples in less than 40 min. These assays are recommended for analysis of restriction digests, amplified DNA, microsatellites, and AFLPs.

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Protein analysis: The Pro260 kit offers the ability to analyze one to ten protein samples (10–260 kD) in approximately 30 min.

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ADDITIONAL RESOURCES: http://www.biorad.com/webroot/web/pdf/lsr/literature/BULLETIN_5761B.pdf

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5868A.pdf

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5909.pdf

 

Integrity control of nucleic acids with a Bioanalyzer 2100 (Agilent)

We offer integrity control of nucleic acids using Agilent 2100 BioAnalyzer that replaces regular gel analysis and offers higher sensitivity. The instrument combines quantitation and quality assessment in a single step. From 1.5 to 2.5 µl of NA sample is required. Kit for measuring RNA is available at CF. 12 samples of RNA or 11 samples of DNA can be measured in one chip, respectively. Researchers can use instrument after training or we can measure your samples for you in form of a service.

Integrity of RNA:

The RIN (RNA Integrity Number) algorithm calculates RNA integrity using a Bayesian learning network that takes into consideration 8 features from the electrophoretic trace, including total RNA ratio (rRNA peaks to total area of the electropherograms), height of the 18S rRNA peak, ratio of the fast migrating RNAs to the total area of the electropherograms, and height of the lower marker (Imbeaud et al. 2005). The output RIN is a number between 1 (completely degraded RNA) and 10 (intact RNA).

 

  • Agilent RNA 6000 Nano Kit for the analysis and quantification of total and mRNA samples of 25 to 500 ng/µL.
  • Agilent RNA 6000 Pico Kit for the analysis and quantification of total and mRNA samples of 50 – 5000 pg/µL.
  • Agilent Small RNA kit for the analysis and quantification of small RNA samples of 6 and 150 nt in size and 50 to 2000 pg/µL.1011

 

Analysis of DNA:

The DNA kits are ideal for automated sizing and quantitation of PCR fragments, restriction digests or fragmented DNA.

  • The DNA 1000, 7500 and 12000 kits offer automated analysis of PCR fragments, restriction digests and next-generation sequencing (NGS) samples. Typical application areas are: amplicon QC in forensic DNA studies, gene expression analysis by RT-PCR, DNA QC for NGS, or detection of genetically modified organisms.
  • The High Sensitivity DNA kit allows analysis of fragmented DNA or DNA libraries sith increased sensitivity – down to 5 pg/µL. Analysis of complex DNA samples – down to 100 pg/µL for fragmented DNA or DNA libraries.121314

 

ADDITIONAL RESOURCES: RNA Integrity Database: http://agilent.cnpg.com/Video/flatFiles/194/

http://www.agilent.com/cs/library/applications/5989-1165EN.pdf

 

Integrity control of nucleic acids with a Fragment analyzer (Advanced Analytical)

We offer measurement of integrity control of your nucleic acids using Fragment Analyzer that is powerful automatic capillary electrophoresis. It covers the full spectrum of DNA, gDNA and RNA quality control. Fragment Analyzer™ does everything from high-resolution analysis to fast DNA separations, across the widest separation range (2 – 40 000 bp). Detection starts from 5 pg/μL. For 12 samples.

Quantitative Analysis Kits: http://aati-us.com/product/fragment-analyzer/reagents

  • DNA/NGS Fragment Analysis
  • Large Fragment Analysis
  • Genomic DNA Analysis
  • RNA Analysis
  • Qualitative/Sizing Analysis Kits

 

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ADDITIONAL RESOURCES: http://aati-us.com/sites/default/files/FA%20Brochure.pdf

 

Reverse transcription

We offer to reverse transcribe your RNA.

Reverse transcription is the synthesis of single-stranded DNA (complementary DNA, or cDNA) using single-stranded RNA as a template, mediated by reverse transcriptases (RTs). The cDNA can be used as a template for amplification by PCR or to generate a cDNA library. In our laboratory, we routinelly use TATAA Grandscript cDNA SuperMix.

Fig. Reverse transcription with random hexamers (from TATAA Hands on course of qPCR)

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ADDITIONAL RESOURCES:

http://www.tataa.com/wp-content/uploads/2012/10/Booklet-TATAA-GrandScript-cDNA-Supermix.pdf

 

Quantitative real-time PCR

Absolute quantification

We offer to perform an absolute quantification for you.

One of the choices for absolute quantification is an intrapolating your unknown samples from a standard curve prepared from standards of known concentration. Concentration is presented as number of copies.

The experiment can also be prepared by automatic pipetting system EpMotion.

ATTENTION: If the standard curve is not precise or is not prepared from the sample similar to the quantified unknown sample, then the concentration obtained from such a standard curve is also not precise or might be wrong. The external calibration curve model has to be thoroughly validated as the accuracy of absolute quantification in real-time RT-PCR depends entirely on the accuracy of the standards. If the good standard is not available, you can try different method, a digital PCR.

Fig:  qPCR KAPA Library Quantification Kit for quantification of NGS libraries

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ADDITIONAL RESOURCES: http://qpcrupdate.com/bustin-2000.pdf

 

Relative quantification

We offer to design and perform qPCR experiment using relative quantification.

Most of qPCR experiments are designed as relative quantification. It means that the changes of expression in a target sample (for example a treated sample) are compared with gene expression of a reference sample (for example an untreated sample). All samples are usually normalized (for example with reference genes). A normalized target sample is compared with a normalized reference sample and as a result, the fold change of gene expression between 2 samples is calculated. The example: Expression of gene X in the treated sample is 5 fold lower than expression of gene X in the untreated sample.

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ADDITIONAL RESOURCES: http://gene-quantification.de/pfaffl-rel-quan-book-ch3.pdf

https://www.youtube.com/watch?v=y8tHiH0BzGY

http://www2.udel.edu/ctcr/sites/udel.edu.ctcr/files/Relative%20Quantification%20Guide.pdf

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5279.pdf

 

 

Selection of appropriate reference genes for your system

The reliability of any relative RT-PCR experiment can be improved by including an invariant endogenous control (reference gene) in the assay to correct for sample to sample variations in RT-PCR efficiency and errors in sample quantification. Ideal reference gene should be stable in ALL conditions of YOUR experiment. Unfortunately there are NO universal reference genes, expressed at constant levels under all conditions in all tissues. That is why candidate reference genes have to be validated for each experimental condition and each type of sample.

We offer to find stable reference genes for your experiment using a reference gene panel. The Reference Gene Panels offer an easy way of screening for reference genes as they contain a set of 12 highly efficient wet lab validated assays that are ready to use. Genes with varying cellular functions and expression levels have been selected to reduce the risk of using co-regulated genes. The primers have been designed to be exon-spanning where possible. There are 3 variants of the panel: for human, mouse and rat.

Figures: Example of Normfinder software search for optimal reference gene. The best option is to use together the first 5 reference genes for normalization.

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ADDITIONAL RESOURCES: http://gene-quantification.de/

http://www.tataa.com/wp-content/uploads/2012/10/Reference-Gene-Panel-Product-Flyer.pdf

 

 

Control of contamination with genomic DNA

Any RNA extraction method co-purifies DNA to some extent, usually around 1%, but can be up to 10%! Genomic DNA (gDNA) contamination is a problem that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR).

How to identify and quantify gDNA contamination in RNA sample:

  • Agarose gel: If there is a prominent band above the 28s peak, it might be a hint of gDNA contamination
  • Check for DNA contamination using specific fluorescent dye present in Qubit dsDNA kit that would recognize DNA from RNA
  • Run a qPCR with DNA-specific primers (i.e. that do work on DNA) on RNA. As RNA is no template for PCR, any signal you get is from contaminating DNA. The control can be done by ValidPrime (gDNA specific primers).
  • Run a qPCR with your reference gene using your RNA instead of cDNA. If you get a signal, it cannot come from RNA, it would come from gDNA.
  • RT- control: minus-reverse transcriptase control (“No Amplification Control” or NAC) in qRT-PCR experiments. Typically, the NAC is a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase. If a product is seen in the NAC in qPCR, it probably indicates that contaminating DNA is present in the sample. An unofficial rule-of-thumb is that you can disregard gDNA contamination (when doing relative quantification) if the gDNA amplifies >5 cycles after the cDNA amplification (i.e. >32 fold less).

How to get rid of genomic DNA:

  • Dnase treatment, either on the column or after RNA purification on the RNA. You should verify after Dnase treatment, that the RNA is free of DNA.
  • RT- control and use the GenEx software: the contribution to Cq from the genomic background can be calculated and the Cq values corrected.
  • Using valid prime control and use the GenEx software: the contribution to Cq from the genomic background can be calculated and the Cq values corrected.
  • Careful primer design: intron needs to be at least 700 bp when using an intron spanning assay; junction assays work too but are more difficult to design (not always possible).

 

Fig. Example of bad quantification of the sample contaminated with genomic DNA.

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We can control the purity of your RNA sample with ValidPrime. ValidPrime™ replaces the need to perform no reverse transcriptase (RT(-)) controls for all samples in your real-time quantitative PCR (qPCR) profiling to test for the presence of genomic DNA (gDNA). Just add the ValidPrime™ assay to the list of assays, and the gDNA control to the list of samples, and run. ValidPrime™ will minimize the amount of control reactions and hence your costs, as well as your efforts.In case there is a gDNA contamination in your RNA sample, the contribution to Cq from the genomic background can be calculated and the Cq values corrected.

ADDITIONAL RESOURCES: http://genexp.ibt.cas.cz/145.pdf

 

Validation of new primers and probes

Providing all relevant assay characteristics can help reviewers to assess the validity of the results. If you buy assays from commercial providers, some characteristics, that are needed for your information or reviewer process for an acceptance of a manuscript, are often missing.

We offer performing all necessary experiments to obtain all relevant information that are needed according to the MIQE guidelines.

  • Optimalization of the temperature profile of the experiment in order to reach the proper anneling temperature of your primers and probes
  • Specificity of the assay: running electrophoretic gel (or microfluidic equivalents) and running melting profile during qPCR (if SYBR green the method of choice)
  • Measuring the efficiency of the assay and finding out the dynamic range of the your experiment
  • Limit of detection or limit of quantification
  • Quality controls: NTC (no template control = contamination control or primer dimer control), gDNA contamination controls (ValidPrime or  RT-), positive or negative controls
  • Optimalization of multiplex

 

Fig. Example of a serial dilution for finding out the efficiency of the primers

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ADDITIONAL RESOURCES: http://genexp.ibt.cas.cz/188.pdf

http://genexp.ibt.cas.cz/118-2.pdf

 

Inhibition control in RT and in qPCR

Contaminants  present  in  samples  are  known  to  inhibit  enzymatic  reactions  and  in  the  context  of  a  reverse-transcription  quantitative  Polymerase  Chain  Reaction  (RT-qPCR)  assay,  inhibitors  are  fully  capable  of  distorting  reported  measurements. The impact to Cq from the inhibition can NOT be recalculated and the Cq values corrected. The experimental sample containing inhibitors should be repurified and reanalyzed for correct biological interpretation. One solution to get out of inhibitors is diluting your sample. However, this approach is not applicable for all inhibitors.

 

The source of inhibition:

  • From sample itself: tissue specific or sample specific inhibition, for example hemoglobin in blood
  • From fixative where is sample fixed, for example FFPE
  • From the isolation technique: for example trizol or chemicals used for stabilization of NA as EDTA, glycogen
  • From reverse transcription: High concentration of RT enzyme is inhibitory or higher concentration of Taq polymerase that can be brought in reaction with higher volume of RNA
  • From interaction of a specific qPCR templet with a specific probe or primer, especially if you have very high concentration of probe interaction

 

We can help you to discover inhibition in your reaction using spikes.

 

The  test  for  inhibition  is  based  on  an  efficient  and  simple  principle: equal amounts  of  spike  are  added  to  all  experimental  samples  and  to  an additional control sample. The control sample is based on nuclease-free water  or  purified  matrix,  which  is  known  to  be  contaminants-free. All samples have the same volume. The experimental and the control sample are then  reverse  transcribed  and  amplified  with  the  Spike  Assay  under  identical conditions.  If  the  Cq  value  is  greater  in  an  experimental  sample than  in  the  control  then  the  analytical  process  of  that  experimental  sample  is  inhibited.  The  magnitude  of  the  difference  between  these  Cq  values reflects the degree of inhibition (from TATAA source).

 

Fig. Example of TATAA RNA spike: A. No inhibition. B. Inhibition in RT. C. Inhibition in qPCR. D. Inhibition both in RT and in qPCR.

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ADDITIONAL RESOURCES: http://www.tataa.com/wp-content/uploads/2012/10/TATAA-Universal-RNA-and-DNA-Spike-Product-Flyer.pdf

 


High-throughput qPCR: BioMark (Fluidigm)

Gene expression: 9216 qPCR rections with GE 96.96 or 2304 qPCR reactions with GE 48.48

High-throughput qPCR System BioMark (Fluidigm) uses integrated fluidic circuits known as dynamic arrays and digital arrays. These innovative products allow scientists to practice tried-and-true techniques, such as TaqMan assays or EvaGreen chemistry, while realizing a previously unachievable throughput. Researchers save on reagents, pipette tips, time, and on the upkeep of expensive liquid-handling robots.

 

Using BioMark you can save:

  • Time – the whole experiment is performed in one day
  • Material –2 ul of sample is enough for 96 gene analysis
  • Mastermix – instead of 46 ml of mastermix during regular qPCR, 240 ul of mastermix is enough
  • Primers and probes – instead of 4.6 ml for all primers, 240 ul is sufficient  (2.5 ul for 1 pair)
  • Tips – instead of 18432 pipet steps,  you will pipette only 196 x
  • Plates – instead of 24 PCR plates (384 wells),  1 Biomark array will do the work
  • Interplate calibration –  if your whole experiment fits into one array, than you do not need interplate calibration
  • Work – we will do the whole experiment for you
  • Money – the whole experiment is cheaper than the regular experiment using 384 well PCR plates

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ADDITIONAL RESOURCES: https://www.fluidigm.com/publications/biomark

 

 

Fluidigm GE arrays available at CF

  • 96.96 Dynamic Array IFC for Gene Expression: combines 96 samples with 96 assays (9216 reactions)
  • 48.48 Dynamic Array IFC for Gene Expression: combines 48 samples with 48 assays (2304 reactions)
  • Flex Six™ IFC for Gene Expression: 6 x reusable chip that combines 12 samples with 12 assays (6x 144 reactions)

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Genotyping: 9216 qPCR reactions with GT 96.96 or 2304 qPCR reactions with GT 48.48

High-throughput qPCR System BioMark (Fluidigm) uses integrated fluidic circuits known as dynamic arrays and digital arrays. These innovative products allow scientists to practice tried-and-true techniques, while realizing a previously unachievable throughput. Researchers save on reagents, pipette tips, time, and on the upkeep of expensive liquid-handling robots.

Using BioMark you can save:

  • Time – the whole experiment is performed in one day
  • Material –2 ul of sample is enough for 96 gene analysis
  • Mastermix – instead of 46 ml of mastermix during regular qPCR, 240 ul of mastermix is enough
  • Primers and probes – instead of 4.6 ml for all primers, 240 ul is sufficient  (2.5 ul for 1 pair)
  • Tips – instead of 18432 pipet steps,  you will pipette only 196 x
  • Plates – instead of 24 PCR plates (384 wells),  1 Biomark array will do the work
  • Interplate calibration –  if your whole experiment fits into one array, than you do not need interplate calibration
  • Work – we will do the whole experiment for you
  • Money – the whole experiment is cheaper than the regular experiment using 384 well PCR plates

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Fluidigm GE arrays available at CF:

  • 96.96 Dynamic Array IFC for Genotyping: combines 96 samples with 96 assays (9216 reactions)
  • 48.48 Dynamic Array IFC for Genotyping: combines 48 samples with 48 assays (2304 reactions)
  • Flex Six™ IFC for Genotyping: 6 x reusable chip that combines 12 samples with 12 assays (6x 144 reactions)

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Available formats of chips:

  • 12.765 Digital Array IFC: for dPCR of 12 samples
  • 48.770 Digital Array IFC: for dPCR of 48 samples

ADDITIONAL RESOURCES: https://www.fluidigm.com/search?query=genotyping

 

Preamplification

Preamplification is specific, exponential amplification of a template, which is used for low concentrated samples and for high-throughput systems as BioMark. Because from one sample is measured amplification of 96 targets, the input concentration of DNA or cDNA needs to be high. The necessary concentration is not possible to reach with reverse transcription without the possibility of inhibition.

We offer preamplification of your samples using specific multiplex PCR that combines all your specific primers in a limited concentration and limited numbers of PCR cycles. We can also offer the validation of preamplification, see the figure.

Fig. Validation of preamplification according to Rusnakova: PloS One 8(8), e69734 (2013).

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ADDITIONAL RESOURCES: http://genexp.ibt.cas.cz/189.pdf

http://genexp.ibt.cas.cz/192.pdf

 

Digital PCR

Droplet digital PCR with QX200 (Bio-Rad) for 8 – 96 samples

The ddPCR System provides absolute quantification of target DNA or RNA molecules for EvaGreen or probe-based digital PCR applications without a need for a standard curve. You can use 2 channels simultaneously (FAM, VIC).

We offer quantification with EvaGreen, probes and NGS libraries. All material is available at CF. Researchers can use instrument by themselves after training.

Droplet digital PCR principle:

  1. Droplet Digital PCR (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. A sample is fractionated into 20,000 droplets.
  2. PCR amplification of the template molecules occurs in each individual droplet.
  3. Each droplet is analyzed or read in a flow cytometer to determine the fraction of PCR-positive droplets in the original sample. So, each positive droplet is calculated (1) and each negative droplet is calculated (0).These data are then analyzed using Poisson statistics to determine the target DNA template concentration in the original sample.

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Aplications of ddPCR:

  • Absolute quantification
  • CNV (Copy number variation)
  • Mutation analysis
  • NGS validation and quantification of libraries
  • Gene expression
  • EvaGreen applications

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Advantages of ddPCR:

  • Absolute quantification — ddPCR technology provides an absolute count of target DNA copies per input sample without the need for running standard curves, making this technique ideal for measurements of target DNA, viral load analysis, and microbial quantification
  • Unparalleled precision — the massive sample partitioning afforded by ddPCR enables the reliable measurement of small fold differences in target DNA sequence copy numbers among samples
  • Increased signal-to-noise ratio — high-copy templates and background are diluted, effectively enriching template concentration in target-positive partitions, allowing for the sensitive detection of rare targets and enabling a ±10% precision in quantification
  • Removal of PCR bias — error rates are reduced by removing the amplification efficiency reliance of qPCR, enabling the detection of small (1.2-fold) differences
  • Simplified quantification — neither calibration standards nor a reference (the ΔΔCq method) is required for absolute quantification
  • Superior partitioning — ddPCR technology yields 20,000 droplets per 20 µl sample, nearly two million partitioned PCR reactions in a 96-well plate, whereas chip-based digital PCR systems produce only hundreds or thousands of partitions. The greater number of partitions yields higher accuracy

 

ADDITIONAL RESOURCES: http://www.bio-rad.com/en-cz/applications-technologies/droplet-digital-pcr-ddpcr-technology#1

https://www.youtube.com/watch?v=9TJy_uegmd4

https://www.thermofisher.com/cz/en/home/life-science/pcr/digital-pcr.html

 

Mikrofluidic digital PCR with BioMark (Fluidigm) for 12 or 48 samples

A sample  in BioMark System is diluted and partitioned into hundreds or even millions of separate reaction chambers so that each contains one or no copies of the sequence of interest. By counting the number of ‘positive’ partitions (in which the sequence is detected) versus ‘negative’ partitions (in which it is not), scientists can determine exactly how many copies of a DNA molecule were in the original sample without need of a standard curve. What is more, you will obtain a real time PCR amplification curves and you can easily disqualify aberrant curves.

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Microfluidic digital PCR principle:

  1. Sample is divided into individual compartments (1 sample into 770 compartments in a digital array)
  2. qPCR amplification of a specific amplicon using primers and probes (VIC and FAM channel)
  3. Positive and negative compartments, respectively, are calculated by software (1 or 0), software calculates with possibilities that there might be more copies of interest in one compartment (Poisson statistics). The target DNA template concentration in the original sample is determined. 

Applications of dPCR:

  • Absolute quantification
  • CNV (Copy number variation)
  • Mutation analysis
  • EvaGreen applications
  • Among other applications, researchers have used digital PCR to distinguish differential expression of alleles, to track which viruses infect individual bacterial cells, to quantify cancer genes in patient specimens and to detect fetal DNA in circulating blood

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All material is available at CF

 

ADDITIONAL RESOURCES: https://www.fluidigm.com/search?query=digital+PCR&applications=digitalPCR

 

Special aplication: Single cell qPCR analysis

It was once assumed that cell populations were homogeneous, but the latest evidence shows that heterogeneity does in fact exist even within small cell populations. Gene expression measurements based on the homogenized cell population are misleading averages and don’t account for the small but critical changes occurring in individual cells. Individual cells can differ dramatically in size, protein levels, and expressed RNA transcripts, and these variations are key to answering previously irresolvable questions in cancer research, stem cell biology, immunology, developmental biology, and neurology (Thermofisher Scientific).

Fig. Analysis of single cells using instrument BioMark (Fluidigm). Heatmap of Cq values in gene expression array.

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Fig. Analysis of single cells using digital PCR QX200 (Bio-Rad).

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ADDITIONAL RESOURCES:

https://www.fluidigm.com/applications/single-cell-analysis

http://www.bio-rad.com/en-cz/applications-technologies/single-cell-analysis-using-digital-pcr

https://www.youtube.com/watch?v=bRK1Dkndiw0

 

 

Automatization

Automatic isolation of nucleic acids with QiaCube (Qiagen) for 12 samples

The Qiagen QiaCube automates the use of standard Qiagen purification kits. Up to twelve samples can be purified at a time. Pre-written purification protocols are freely downloadable from Qiagen and must be uploaded on to the QiaCube before use (http://www.qiagen.com/qiacube/standard/search.aspx). The pre-written protocols can be modified according to user needs. The instrument gives very repetitive purification results. The automation of  NA extraction has the advantage of a standardized sample treatment and avoidance of error during routine sample handling and contamination due to intermediate process.

Bring your samples and obtain isolated nucleic acids back. Or get training and isolate with QiaCube on your own. Tips for the system are available at CF.

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ADDITIONAL RESOURCES: http://www.marshall.edu/forensics/files/LOWNEYJESSICA_RESEARCH_PAPER.pdf

http://www.quantum.ee/failid/Upload/pdf/1081139_fold-qiacube_0214.pdf

 

Automatic pipetting and preparation of dilution series with epMotion P5073 (Eppendorf)

With epMotion automated pipetting systems, you will see things in a new light. All your routine pipetting tasks, whether small or large, will be automated with more precision and safety than you ever experienced with manual pipetting

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ADDITIONAL RESOURCES: http://www.eppendorf.com/int/img/itscalledepmotion/epmotion.pdf

http://www.artel-usa.com/wp-content/uploads/2013/09/12A6346_Evaluation-of-the-Eppendorf-epMotion-Pipetting-Tools-using-the-Artel-MVS.pdf

VIDEOS: https://www.youtube.com/watch?v=vgJdySaNPXY

https://www.youtube.com/watch?v=REVXumkaYrg&nohtml5=False

https://www.youtube.com/watch?v=Z7ZjgPwl2Yo&nohtml5=False

https://www.youtube.com/watch?v=VfkESROkFRY&nohtml5=False

https://www.youtube.com/watch?v=nLpOxGxOGZ0&nohtml5=False

https://www.youtube.com/watch?v=9GpzSGnWhA0&nohtml5=False

 

 

 

 

References and Publications

CONNECTION TO INTERNATIONAL RESEARCH INFRASTRUCTURES OR CONSORTIA

  • SPIDIA: EU consortium for the standardisation and improvement of pre-analytical procedures for in-vitro diagnostics
  • cooperation with companies: TATAA Biocenter, Bio-Rad, Eppendorf, Qiagen, Roche
  • cooperation with universities: Technische Universität München, Germany; Tun Abdul Razak Research Centre London, England; Erasmus University Rotterdam, Netherlands; The University of Queensland, Brisbane, Australia

 

SELECTED REFERENCES ON METHODOLOGY

  • The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Clin Chem. 2009 Apr;55(4):611-22.
  • Gene expression profiling–Clusters of possibilities. Bergkvist A, Rusnakova V, Sindelka R, Garda JM, Sjögreen B, Lindh D, Forootan A, Kubista M. Methods. 2010 Apr;50(4):323-35.
  • Statistical aspects of quantitative real-time PCR experiment design. Kitchen RR, Kubista M, Tichopad A. Methods. 2010 Apr;50(4):231-6.
  • Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime. Laurell H, Iacovoni JS, Abot A, Svec D, Maoret JJ, Arnal JF, Kubista M. Nucleic Acids Res. 2012 Apr;40(7).
  • The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA. Clin Chem. 2013 Jun;59(6):892-902.
  • RT-qPCR work-flow for single-cell data analysis. Ståhlberg A, Rusnakova V, Forootan A, Anderova M, Kubista M. Methods. 2013 Jan;59(1):80-8.
  • The workflow of single-cell expression profiling using quantitative real-time PCR. Ståhlberg A, Kubista M. Expert Rev Mol Diagn. 2014 Apr;14(3):323-31.
  • Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples.Zhang H, Korenková V, Sjöback R, Švec D, Björkman J, Kruhøffer M, Verderio P, Pizzamiglio S, Ciniselli CM, Wyrich R, Oelmueller U, Kubista M, Lindahl T, Lönneborg A, Rian E. PLoS One. 2014 Nov 4;9(11).
  • Pre-amplification in the context of high-throughput qPCR gene expression experiment. Korenková V, Scott J, Novosadová V, Jindřichová M, Langerová L, Švec D, Šídová M, Sjöback R. BMC Mol Biol. 2015 Mar 11.

 

PUBLICATIONS WITH COOPERATION OF CF

2015

  • Pre-amplification in the context of high-throughput qPCR gene expression experiment. Korenková V, Scott J, Novosadová V, Jindřichová M, Langerová L, Švec D, Šídová M, Sjöback R. BMC Mol Biol. 2015 Mar 11;16:5. doi: 10.1186/s12867-015-0033-9.
  • Post-treatment recovery of suboptimal DNA repair capacity and gene expression levels in colorectal cancer patients. Slyskova J, Cordero F, Pardini B, Korenkova V, Vymetalkova V, Bielik L, Vodickova L, Pitule P, Liska V, Matejka VM, Levy M, Buchler T, Kubista M, Naccarati A, Vodicka P. Mol Carcinog. 2015 Sep;54(9):769-78. doi: 10.1002/mc.22141. Epub 2014 Mar 3.

2014

  • SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses. Malentacchi F, Pazzagli M, Simi L, Orlando C, Wyrich R, Günther K, Verderio P, Pizzamiglio S, Ciniselli CM, Zhang H, Korenková V, Rainen L, Bar T, Kubista M, Gelmini S. PLoS One. 2014 Nov 10;9(11):e112293. doi: 10.1371/journal.pone.0112293. eCollection 2014.
  • Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples. Zhang H, Korenková V, Sjöback R, Švec D, Björkman J, Kruhøffer M, Verderio P, Pizzamiglio S, Ciniselli CM, Wyrich R, Oelmueller U, Kubista M, Lindahl T, Lönneborg A, Rian E. PLoS One. 2014 Nov 4;9(11):e111644. doi: 10.1371/journal.pone.0111644. eCollection 2014.
  • Molecular characteristics of mismatch repair genes in sporadic colorectal tumors in Czech patients. Vymetalkova VP, Slyskova J, Korenkova V, Bielik L, Langerova L, Prochazka P, Rejhova A, Schwarzova L, Pardini B, Naccarati A, Vodicka P. BMC Med Genet. 2014 Jan 31;15:17. doi: 10.1186/1471-2350-15-17.
  • Effect of zearalenone on reproductive parameters and expression of selected testicular genes in mice. Zatecka E, Ded L, Elzeinova F, Kubatova A, Dorosh A, Margaryan H, Dostalova P, Korenkova V, Hoskova K, Peknicova J. Reprod Toxicol. 2014 Jun;45:20-30. doi: 10.1016/j.reprotox.2014.01.003. Epub 2014 Jan 9.

2013

  • Evaluation of tumor suppressor gene expressions and aberrant methylation in the colon of cancer-induced rats: a pilot study. Polakova Vymetalkova V, Vannucci L, Korenkova V, Prochazka P, Slyskova J, Vodickova L, Rusnakova V, Bielik L, Burocziova M, Rossmann P, Vodicka P. Mol Biol Rep. 2013 Oct;40(10):5921-9. doi: 10.1007/s11033-013-2699-8. Epub 2013 Sep 25.
  • Association of obesity susceptibility gene variants with metabolic syndrome and related traits in 1,443 Czech adolescents. Dušátková L, Zamrazilová H, Sedláčková B, Včelák J, Hlavatý P, Aldhoon Hainerová I, Korenková V, Bradnová O, Bendlová B, Kunešová M, Hainer V. Folia Biol (Praha). 2013;59(3):123-33.
  • Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens. Sorg D, Danowski K, Korenkova V, Rusnakova V, Küffner R, Zimmer R, Meyer HH, Kliem H. Animal. 2013 May;7(5):799-805. doi: 10.1017/S1751731112002315. Epub 2012 Dec 11.