Mar 21, 2025

Public workspaceGenotyping the SNP rs1143634 of the gene Interleukin-1β with Real-Time Polymerase Chain Reaction (PCR) in dengue patients 

DOI
dx.doi.org/10.17504/protocols.io.6qpvr9ow3vmk/v1
  • Raquel da Silva Carvalho1,
  • Vanessa Rafaela Milhomen Cruz Leite2,
  • Jéssica Barletto de Sousa Barros1,
  • Irmtraut Araci Hoffmann Pfrimer1
  • 1Postgraduate in Environmental and Health Sciences, Pontifical Catholic University of Goiás (PUC-GO), Center for Immunological Studies and Research (NEPY), Goiânia, Goiás;
  • 2Laboratory of Genetics and Molecular Biology - Institute of Biological Sciences II - Federal University of Goiás, Goiânia, Goiás
  • Jéssica Barletto de Sousa Barros: These authors contributed equally to this work;
  • Irmtraut Araci Hoffmann Pfrimer: These authors contributed equally to this work
Raquel da Silva Carvalho
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr9ow3vmk/v1
Protocol Citation: Raquel da Silva Carvalho, Vanessa Rafaela Milhomen Cruz Leite, Jéssica Barletto de Sousa Barros, Irmtraut Araci Hoffmann Pfrimer 2025. Genotyping the SNP rs1143634 of the gene Interleukin-1β with Real-Time Polymerase Chain Reaction (PCR) in dengue patients . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr9ow3vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 15, 2024
Last Modified: March 21, 2025
Protocol Integer ID: 112134
Keywords: Dengue; Genotyping; IL-1β, single nucleotide polymorphism
Abstract
Interleukin 1 (IL-1) plays a role in regulating inflammatory responses and is crucial for both innate and adaptive immunity. The secretion of IL-1β can influence various biochemical, physiological and pathological functions in the body, ranging from polarization towards a Th1 response and intensification of pro-inflammatory mediators in the acute phase, to stimulation of chronic inflammation and carcinogenic potential in some cases. In addition, dengue cases are alarmingly high every year, reaching thousands of cases in Brazil and Latin America. The increase in severe cases of dengue may be related to genetic factors, such as the SNP rs1143634 (G>A) in the interleukin 1- Beta (IL-1β) gene. Although synonymous, this SNP can influence the immune response, potentially aggravating the infection.
Materials
  1. PCR 96- Well Plate (DNASE FREE)
  2. SEALANT
  3. PIPETTE (V°: 2/ 10/ 20 MICROLITERS)
  4. TIP (V°: 2/ 10/ 20 Microliters DNASE FREE with Filter )
  5. DNA SAMPLE - Extracted and Quantified
  6. MASTER MIX (THERMO FISHER)     
  7. TAQMAN GENOTYPING ASSAY (THERMO FISHER)
  8. NUCLEASE-FREE WATER
INITIAL PREPARATION
INITIAL PREPARATION
Before starting
During the extraction of human DNA, attention must be paid to possible sources of contamination, as well as to the manufacturer's recommendations.
Figure 1: Extraction of human DNA by PureLink‱ Genomic DNA Mini Kit (Invitrogen), for future genotype detection.

PCR
GENERAL ASPECTS
  1. Definition: The polymerase chain reaction (PCR) is a technique used to detect RNA and DNA, with the test focusing on DNA. This technique, also known as quantitative PCR or quantitative real-time PCR, is a method of simultaneously quantifying and amplifying DNA. Real-time PCR is based on the detection and quantification of a fluorescent compound (during the early stages of PCR), based on the basic principle that the significant increase in the amount of PCR product correlates with the initial amount of the control. This technique requires only 3 picograms of material, which is around 1000 times less genetic material than conventional assays.
  2. Objective: Quantify and amplify a specific DNA sequence (SNP IL-1β rs1143634), identifying the presence of the SNP.
Download 20240603_162117 (1).mp420240603_162117 (1).mp49.3MB
PERSONAL PROTECTIVE EQUIPMENT
  • Nitrile gloves
  • Disposable cloak/sleeved jacket
  • Goggles
  • Surgical mask
  • Gown

REAGENTS NEEDED
PCR 96- Well Plate (DNA'SE FREE)
  1. SEALANT
  2. PIPETTE (V°: 2/ 10/ 20 MICROLITERS)
  3. TIP (V°: 2/ 10/ 20 Microliters DNA'SE FREE with Filter )
  4. DNA SAMPLE - Extracted and Quantified
  5. MASTER MIX (THERMO FISHER)     
  6. TAQMAN GENOTYPING ASSAY C___9546517_10 (THERMO FISHER Scientific)
  7. NUCLEASE-FREE WATER
AB
REAGENTS VOLUME PER REACTION
MASTERMIX 10ul
TAQMAN PROBE 1ul
GENOMIC DNA 4ul
NUCLEASE-FREE WATER 5ul
TOTAL VOLUME 20 ul
Table 1: Volume of reagents per reaction for Real-Time Polymerase Chain Reaction to detect genotypes.
Pipetting
Mix
CYCLING PANEL
PCR was carried out using Roche's LightCycler 480 II thermal cycler.

Roche's LightCycler 480 II thermal cycler

ABCD
STAGE TEMPERATURE TIME CYCLE
PRE-READING 60 C° 30s 1
ENZYME ACTIVATION 90 C° 5m 40
DENATURATION 60 C° 30s 1
RINGING/EXTENSION 60 C° 30s 1
POST-READING 60 C° 30s 1
PCR
Critical
DESCRIPTION OF THE STEPS
DESCRIPTION OF THE STEPS
Equipment: 96-well plate
Functions:  To carry out the reaction.
Note: The plate has a specific barcode, so it cannot be reused.
Mix
Process: Mix each solution well and centrifuge or Multi Mix (Vortex) for up to 1 minute to remove air bubbles.
Pipetting
Equipment: 96-well optical plate
Functions: Mix the volumes of each reagent in each well of the plate up to the pre-set volume.
Equipment: Close the plate with the sealant, then centrifuge for another 5 minutes to remove the air bubbles until well homogenized.
Centrifigation
Equipment: Store the plate with the samples for up to 72 hours.
Analyze
ACTIONS IN CASES OF NON-COMPLIANCE
ACTIONS IN CASES OF NON-COMPLIANCE
  • After identifying the non-conformity, describe it in detail in the laboratory log. Describe the errors and the actions taken to correct them.
Analyze
  • Call technical assistance if the equipment is faulty or out of calibration.
INTERPRETING GENOTYPING RESULTS
INTERPRETING GENOTYPING RESULTS
After real-time PCR, when the curves of each sample are analyzed, the genotypes are recognized as Wild (GG), being those that have not mutated, Heterozygous (GA), with an exchange of nitrogenous bases in one chromosome and finally Mutant (AA) with total exchange. In the Roche thermal cycler they are identified by color, Wild (GG) by blue, Heterozygous (GA) by red and Mutant (AA) by green.
This data will be analyzed by statistics using the R studio software.
Figure 3: Result after real-time PCR.


PCR
Analyze
Computational step
Protocol references
1- OLIVEIRA, R: PCR em tempo real: métodos e aplicações. Universidade de Aveiro, 2010. http://hdl.handle.net/10773/7230

2- MENEZES, M;LIMA, L; MARTINELLO, F: Laboratory diagnosis of SARS-CoV-2 by real time reverse transcriptase-polymerase chain reaction (RT-PCR). Universidade Federal de Santa Catarina (UFSC). 2020 DOI: 10.21877/2448-3877.20200006

3-OLIVEIRA M;WATANABE, A; CESAR, D; CANDIDO, J; LIMA, N; MOREIRA, O:TESTES DIAGNÓSTICOS PARA O SARS-COV-2: UMA REFLEXÃO CRÍTICA. Quím Nova [Internet]. 2022Jun;45(6):760–6. Available from: https://doi.org/10.21577/0100-4042.20170895