A primer pair for the SNP01 (G > A) was designed as already described to amplify the small fragment containing the target SNP of c.-81-176G > A, which was further subjected to HRM analysis (Supplemental Table 1 ). HRM was performed on sequencing-verified samples in order to determine the Tm and characterize of the melting curve profiles of different genotypes (GG and AG). PCR reactions were performed on StepOne real-time PCR detection system (Applied Biosystems®, USA). All samples were amplified in duplicate, and each run contained a non-template control (NTC) and two type individuals as the heterozygous and homozygous genotypes. The 20 μL reaction volume included 1 × Fast EvaGreen® Master Mix (Biotium Inc., USA), 20 ng DNA, 0,1 μM each primer and sterile water. The cycling conditions were an initial denaturation at 95 °C for 10 min, 40 cycles of 15 s at 95 °C, 1 min at 60 °C. For HRM analysis, the PCR product was denatured by rising temperature to 95 °C at 4.8 °C/s and was then cool down to 55 °C at 2.5 °C/s for hybridization. The melting curve was acquired by increasing the temperature from 55 °C to 95 °C at a ramp rate of 4.8 °C/s with 25 acquisitions per degree of temperature. The HRM Software v2.0.1 (Applied Biosystems®, USA) was used to auto group the melt curve, and each genotype produced easily discriminated melt curves (amplitude and/or shape of the curves).
Fast evagreen master mix
Manufactured by Biotium
Sourced in United States
Fast EvaGreen® Master Mix is a ready-to-use solution designed for real-time PCR applications. It contains the necessary components, including EvaGreen® dye, for efficient DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Stepone real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Superscript 3 platinum one step qrt pcr system, by Thermo Fisher Scientific (1 mentions)
Steponeplus thermocycler, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
β actin, by GeneCopoeia (1 mentions)
Lightcycler 2, by Roche (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
4 protocols using fast evagreen master mix
1
High-Resolution Melting Analysis for SNP Genotyping
2
Quantitative Real-Time PCR Analysis
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Total RNA was used to make cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara). qPCR was performed on an ABI 7500 instrument using the Fast EvaGreen Master Mix (Biotium). Thermal cycling consisted of an initial step at 95°C for 10 min followed by 40 cycles at 95°C for 30s and 62°C for 30s. The qPCR measurements were performed in triplicate for each cDNA sample and gene expression was quantified relative to either RPS18, ACTB, or GAPDH expression using the 2-ΔCt method. Data were analyzed using SAS 8.0.
3
Cytokine expression analysis in Hec1b cells
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For those strains that promoted morphological alterations in Hec1b cells were performed analysis of the cytokines (IL10 and TNFα) production. Total RNA was extracted using the Trizol Reagent (Invitrogen, Calsbag, CA, USA). RNA yield was estimated by Nanodrop (Thermo Scientific). A minimum amount of 0.2 ng of the RNA was submitted to reverse transcription followed by qRT PCR. Real-time PCR primers are listed in Table 2 and the reaction was performed using the StepONE Plus thermocycler (Applied Biosystems). Each 10 μL reaction contained 400 nM of each primer, 5 μL Master Mix and 1/60 000 Fast EVA Green Master Mix (both from Biotium), 0.25 uL of Super Script III Platinum One-Step qRT-PCR System (Invitrogen Calsbad, CA, USA) and the recommendations for cDNA production described in the kit were followed. Reactions were repeated on three different biological replicates using as endogenous control the GAPDH gene and fold expression changes were averaged. The data was analyzed by the comparative Ct method revised in (Schmittgen and Livak 2008 (link)).
4
Quantitative Analysis of HMMR and CD44 Expression
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