Gene Core – Quantitative and digital PCR

Gene Core – Quantitative and digital PCR

Ing. Eva Rohlová

Ing. Eva Rohlová — Laboratory head

About us

BIOCEV Gene Core – the best equipped core facility and service provider in the field of gene expression in Central Europe. 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.

We emphasise quality control, which is often neglected. Effective QC is based on the use of molecular tools to control contamination (RNA/DNA spikes), genomic background (ValidPrime) and quality of RNA (ΔAmp, RIN). We also take part in development of these methods to facilitate analysis of gene expression starting from bulk samples, down to the level of individual cells (direct lysis).

In addition to conventional qPCR analysis, we focus on single cells expression profiling and multi-analyte approach. Analysis of DNA/RNA/protein in parallel in one sample even on the single cell level provides comprehensive tool to map gene expression and characterizes types of cells and to determine the degree of differentiation and to study the pathological condition.

Currently we offer assistance with library preparations and experimental design of RNA-Seq experiments, which are key preconditions for a successful project. And also new Two-Tailed PCR for ultrasensitive analysis of microRNAs. Based on an innovative novel design with a RT primer sensing the microRNA using two connected hemi-probes exceeding sensitivity and superior specificity is achieved.


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 – 2.2

Integrity and quantity control of nucleic acids


How to interpret RNA peaks:

What contamination of your NA looks like:

Importance of 260/230 and 260/280 ratios:

Troubleshooting for 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.


  • 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.

Quantity control of nucleic acids with a fluorospektorphotometer Quibit 2.0


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.

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.

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




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.

Integrity of RNA measurment


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.

Analysis of DNA

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.



RNA Integrity Database:


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.

Integrity control of nucleid acids

Quantitative Analysis Kits:

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



Our Equipment

QX200 droplet digital PCR system (Bio-Rad)

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.



Qubit® 2.0 Fluorometer (life Technologies)

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.



GentleMACS™ Dissociator (MACS Miltenyi Biotec)

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.




Fragment Analyzer (Agilent)

Fragment Analyzer (Agilent)

Fragment Analyzer (Agilent) 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.




CFX96 Real Time PCR Instrument (Bio-Rad)

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





epMotion P5073 Automated Pipetting System (Eppendorf)

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




C1000 Thermal Cycler (Bio-Rad)

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)

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.



  • 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.



BioMark qPCR System (Fluidigm)

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 wavelenght 500-550 nm, 540-600 nm
Examples of supported probe types FAM-MGB, VIC-MGB, FAM-TAMRA, FAM-non fluorescent quencher


  • 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.



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TissueLyser LT (Qiagen)

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.







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


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.

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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.


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.