Source: https://pixabay.com/illustrations/dna-analysis-research-3539309/
The Real-Time polymerase chain reaction (RT-PCR) equipment in Centro de Química da Madeira (CQM, University of Madeira) supports research at CQM used as a routine tool for the evaluation of gene expression by CQM researchers from national R&D projects (Post-Doc, PhDs, and Biochemistry (BSc and MSc) and Nanochemistry and Nanomaterials (MSc) students). However, CQM RT-PCR is also open to external services (private and other public institutions).
Submit sample here (exclusively for CQM members) colocar link para submissão de formulário
After the form submission, you will receive an email in your mailbox confirming successful sample registration and the filled form.
Schedule Real-time PCR analysis
If you are not a CQM member and would like to perform a PCR analysis, please contact us via and check the pricing list below.
Polymerase Chain Reaction (PCR)
PCR is a molecular biology technique that allows the amplification of specific DNA sequences, resulting in several copies of the target DNA (or RNA) segments. This principle relies on an initial mixture of the target template DNA, specific primers, nucleotides (dNTPs), ions, and polymerase enzymes that, upon several heating and cooling cycles, promote a three-step cycle of denaturation, annealing, and elongation of the primers.
Denaturation is the first step and involves heating the PCR mixture to a high temperature (approximately 94-98°C), causing the DNA template to denature into two single-stranded chains. The second step, annealing, occurs at lower temperatures (approximately 55-70°C), allowing the short primers to bind to their complementary sequence on each single-stranded DNA template. Finally, in the last step, elongation, the temperature is increased (approximately 68-72°C), promoting the polymerase enzyme to extend the primer strand according to the single-stranded DNA template sequence and synthesise a complementary strand. The end product is a double strand composed of a DNA template chain and a complementary elongated primer. The process is then repeated, usually for up to 40 cycles, resulting in an exponential increase in the number of copies of the target DNA sequence after each cycle.
Advanced molecular approaches have been developed, including real-time PCR, reverse transcription PCR (RT-PCR), and quantitative polymerase chain reaction (qPCR). Real-time PCR takes advantage of fluorescent molecules, making real-time detection of the amplification products possible by increasing the fluorescent signal. Simultaneously, this provides quantitative information (qPCR) on the initial amount of target DNA in the sample. In contrast, RT-PCR allows for the amplification of specific RNA sequences, with a preliminary step where the template DNA is converted into a complementary DNA (cDNA) sequence using reverse transcriptase. This cDNA is then used in the following PCR amplification steps.
PCR is a very fast, sensitive, and specific technique, and its application in modern research includes several scientific fields, such as genetics for genetic mutations and genotyping purposes, diagnostics for viral and bacterial infection detection and treatment, biotechnology to study gene and drug responses for personalised medicine, and forensics, among others.
AZURE CIELO 6 Real-time PCR system (Azure Biosystems, Inc)
The equipment was installed in CQM/UMa under its strategic funding project reference: UIDP/00674/2020.
Specifications
| Part number | AIQ060 |
| Product description | 96-well Real-Time PCR instrument with 10.2” touchscreen interface, 6 dye channel filters |
| Sample capacity (wells) | 96 (plates: low-profile, on-skirted, semi-skirted; tubes: low-profile tubes, individual, strips, flat cap; white, frosted or clear and sealed with optical grade film) |
| Reaction volume | 5–150µL (10–50 µL recommended) |
| Excitation source | LED |
| Detection channels | 6 |
| Multiplexing | Up to 6 targets |
| Thermal element | Peltier |
| Max. block ramp rate | 6°C/sec |
| Avg. sample ramp rate | 4°C/sec |
| Temperature uniformity | ±0.2°C |
| Temperature accuracy | ±0.1°C |
| Dye compatibility | SYBR Green, EvaGreen, FAM, VIC, JOE, HEX, CAL Fluor 540, CAL Fluor Orange 560, ROX, TAMRA, TEX615, Quasar 670, CAL Fluor Red 610, Cy5, LIZ, Mustang Purple, Cy5.5, Quasar 705 |
| Custom dye/chemistry | YES |
| Chemistry capability | Fast/Standard |
| Detection sensitivity | 1 copy |
| Sensitivity | Detect differences as small as 1.5-fold in target quantities in single plex reactions |
| Connectivity | USB, Wi-Fi, Ethernet |
Fluorescence channels and dye/probes compatibility
| Channel 1 | Channel 2 |
| Ex 475 ± 14 | Ex 527 ± 10 |
| Em 524 ± 12 | Em 565 ± 12 |
| Dye SYBR™ Green, EvaGreen™, FAM™ | Dye VIC®, HEX™, JOE™, CAL Fluor® 540, CAL Fluor®, Orange 560 |
| Channel 3 | Channel 4 |
| Ex 572 ± 7.5 | Ex 537 ± 13 |
| Em 623 ± 12 | Em 583 ± 11 |
| Dye TAMRA™, Cy3 | Dye ROX™, TEX®615, CAL Fluor® Red 610 |
| Channel 5 | Channel 6 |
| Ex 623 ± 12 | Ex 655 ± 7.5 |
| Em 676 ± 18.5 | Em 711 ± 12.5 |
| Dye CY®5, Quasar®670, Liz®, Mustang Purple® | Dye CY® 5.5, Quasar 705 |
Applications
Gene expression: functional gene products (usually proteins) resulting from a specific gene being transcribed or “expressed”, as a result of a response of the cell to its environment. After DNA transcription, RNA must be transcribed into a functional protein. Because of the involvement of gene expression in disease processes, several molecular methods have been developed to detect very small amounts of DNA and RNA. Using fluorescence technology, such as the most commonly used markers TaqMan and SYBR green, small changes in gene expression can be detected and quantified.
Genotyping involves identifying differences in genetic information between individuals, tracking strains in crop and plant breeding, and tracking the presence of a transgene in transgenic animal models. It is also used in forensics to assess the probability that a specific individual is the source of a DNA sample and in kinship analysis, such as paternity testing. It is often used to detect known variations in specific genes or identify new ones that may be associated with a specific phenotype through copy number variations, insertions, deletions, or single base pairs (SNPs).
Library quantification: Next-generation sequencing (NGS) is a large-scale DNA sequencing technology used to determine DNA and RNA sequences. It aids researchers in studying genetic variations related to diseases, identifying pathogens, and studying tumour subclones. To perform NGS, a sequencing library is created by randomly fragmenting genetic material and adding adapter sequences. The quantification of libraries is essential to ensure equal representation before pooling and optimal dilution for sequencing. qPCR is the recommended method for library quantification owing to its precision, sensitivity, and ability to quantify amplifiable molecules.
Pathogen detection: Detection of the presence of pathogens is important. In recent years, molecular methods, such as real-time PCR (qPCR), have become increasingly popular for pathogen detection because they can offer accurate detection without the need for much time and resources. As many pathogen genomes have been sequenced, qPCR primers specific for groups of pathogens or even a specific pathogen can allow qPCR to serve as a diagnostic tool. Time is often critical for pathogen detection, particularly with regard to patient outcomes. qPCR allows for the detection, quantification, and typing of pathogens much faster than the traditional culture methods.
Multiplex qPCR: refers to having numerous primer sets within a single PCR mixture to evaluate multiple targets in one sample. To differentiate between the products, target-specific primers with different fluorophore labels were used, and each amplification product was detected using a different fluorescent channel. One benefit of multiplexing is that multiple probes can be used in one sample. Multiplexing also ensures minimal crosstalk between the fluorescent dye/probe owing to similar excitation or emission wavelengths. Traditionally, end-point multiplex PCR analyzes the final gene products by gel electrophoresis. This required the final PCR products to be different sizes to be visibly distinguishable from each other on the gel. Using different fluorescent probes eliminates the need for different sizes of PCR products because instead of visually evaluating the size difference, the amount of fluorescence is analysed. Because each PCR product is associated with a specific fluorescent probe, the amount of fluorescence detected in a sample signifies that the target PCR product was present and to what degree.
Real-time PCR Services
User fees are intended to cover CQM real-time PCR expenses, which include analysis, consumables, parts, and maintenance.
Choose the class that is best suited to your case or consult us using the address:
The price of the services provided is divided into three classes:
- CQM Research groups
- FCT National Centers and National Public Laboratories
- Companies and private Research Centers
Experimental plan of real-time PCR (gene expression)
1 – RNA extraction from samples using the total RNA isolation kit and/or TRIzol method, quantification, and quality assessment using a NanoDrop spectrophotometre.
2 – Reverse transcription (cDNA synthesis), quantification, and quality assessment.
3 – PCR primers. Each primer set is validated for specificity by qPCR and dissociation curve analysis (SYBR green-based qPCR).
4 – Set up the real-time RT-qPCR reactions using the purified cDNA and run in triplicate qPCR alongside controls (ctrl) and housekeeping (hk) gene, SYBR green-based qPCR.
5 – Data analysis. The average value is normalised to the hk gene selected by the applicant for each sample. To evaluate the results, the ΔΔCt or ΔΔCq method is used to compare changes in gene expression between the controls and samples.
Data analysis is performed using the qRT-PCR data analysis software. The primer sequences, raw Ct values, and analysed data, including all fold-change information, are included in the report.
6 – Use of CQM equipment for PCR runs, reagents not included. Samples previously prepared by the user; ready-to-run.
| Services | Timeline | Pricing (€/sample)* | ||
| Class 1 | Class 2 | Class 3 | ||
| #1 | 2 days | 8.50 | 42.50 | 85 |
| #2 | 1 day | 6.50 | 32.5 | 65 |
| #3 | 2-4 weeks | 30 | 60 | 100 |
| #4 | 3 days | 30 | 60 | 100 |
| #5 | 1 day | 50 | 80 | 120 |
| #6 PCR run
(€/well, reagents not included) |
0.5 | 2.5 | 5 | |
| *Prices for services #1-4 include the cost of material and reagents.
Service #4 prices are per sample per gene of interest, including ctrl and one hk (in triplicate) Service prices are volume-dependent. Prices valid from September 2023; VAT not included, please add tax at the rate of 22% |
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