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.


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



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.



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.




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).

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.

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



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



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).



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.

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.


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.