Gene Core – Quantitative and digital PCR

Gene Core – Quantitative and digital PCR

Ing. Lucie Langerová

Ing. Lucie Langerová — 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.

https://academic.oup.com/nar/article/3958703/Two-tailed-RT-qPCR-a-novel-method-for-highly

News

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.

Equipment

Our Equipment

The AVISO CellCelector (Automated Lab Solutions)

The AVISO CellCelector (Automated Lab Solutions)

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)

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)

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 

VIDEO 

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.

ADDITIONAL INFORMATION 

VIDEO 

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.

ADDITIONAL INFORMATION 

VIDEO 

QIAcube (Qiagen)

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)

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)

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 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 

VIDEO

Experion Automated Electrophoresis Station (Bio-Rad)

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 

VIDEO 

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

 

 

ADDITIONAL INFORMATION 

VIDEO 

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

ADDITIONAL INFORMATION 

VIDEO 

VIDEO2 

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.

 

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 

VIDEO 

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



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)

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 

VIDEO1 

VIDEO2 

Publications

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.