The expression of HWP1, SAP4, ALS1, ALS3, HYR1, BCR1, EFG1, ECE1, and CPH1 genes was evaluated using real-time PCR after treatment with quinalizarin. The C. albicans cells were exposed to anthraquinone at the concentration of MIC/2 (4 µg/mL) or 1% DMSO (control) during propagation in liquid culture in Spider medium with 10% fetal bovine serum. After 10-h incubation at 37°C, the yeast cells were collected, and total RNA was extracted with the YeaStar RNA kit (Zymo Research, USA) according to the manufacturer’s instructions. cDNA was then synthesized using a Smart First Strand cDNA Synthesis Kit (EurX, Poland) following the manufacturer’s instructions. For PCR detection of transcripts, we used TaqMan gene expression assays (Lot: 170255, designed by the manufacturer, ThermoFisher Scientific, England) and the Fast Probe qPCR Master Mix (EurX, Poland). The cDNA samples were pre-treated with uracil-N-glycosylase at 37°C for 2 min to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95°C for 3 min, followed by 40 amplification cycles with denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s using QuantStudio3 (Applied Biosystems). The relative level of expression of the analyzed genes was calculated with the 2-(∆∆Ct) method using ACT1 as a reference gene (43 (link)).
Smart first strand cdna synthesis kit
Manufactured by EURx
Sourced in Poland
The Smart First Strand cDNA Synthesis Kit is a tool for the reverse transcription of RNA into complementary DNA (cDNA). It provides the necessary reagents and instructions to convert RNA samples into single-stranded cDNA molecules, which can then be used for various downstream applications, such as PCR amplification, gene expression analysis, or library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Yeastar rna kit, by Zymo Research (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Fast probe qpcr master mix, by EURx (1 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Forward and reverse primer, by Merck Group (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Sg qpcr master mix, by EURx (1 mentions)
K2po4, by Merck Group (1 mentions)
Probe qpcr master mix, by EURx (1 mentions)
Rotor gene 6000, by Qiagen (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Oligonucleotide primer, by Merck Group (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Rna extracol, by EURx (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
7 protocols using smart first strand cdna synthesis kit
1
Quinalizarin Modulates Candida Gene Expression
2
Real-time qPCR for Pluripotency Markers
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The cDNA was prepared using the smART First strand cDNA Synthesis kit (Eurx, Gdańsk, Poland, cat.no. E0804). The cDNA was amplified by real time qPCR with the primers shown in Table 2 . Primers for SSEA-1, CVH and DAZL were designed using Primer3 (v.0.4.1) [40 (link)]. The reactions were performed in a 20-µL volume containing 10 ng cDNA; 0.25U UNG (uracil-N-glycosylase); and 15 pmol of each forward and reverse amplification primer in 1× SG qPCR master mix (Eurx, Gdańsk, Poland, E0401). Thermocycling conditions for real time qPCR were as follows: 1 cycle for UNG pre-treatment at 50 °C for 2 min, 1 cycle for initial denaturation at 95 °C for 10 min; and 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Melting-curve profiles were analyzed for all amplicons using the following thermal conditions: 95 °C for 5 s, 70 °C for 1 min, and then a gradual temperature increase to 95 °C at a ramp rate of 0.11 °C/s. Amplification was performed in Roche Light Cycler 480 v. II real-time system (Roche, Basel, Switzerland).
3
Validating Microarray Results by RT-qPCR
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To validate the microarray results the RT-qPCR was performed in triplicate using QUANTUM EvaGreen® PCR Kit (Syngen Biotech) according to the manufacturer’s instructions and a Mic Real-Time Cycler (Bio Molecular Systems). The primers used (Additional file 16 ) were based on qPrimerDB (version 1.2) data. Primer specificity was verified by melting curve analysis. Each 20 μl reaction mixture contained 4 μl of QUANTUM EvaGreen® PCR Kit mix, two primers (4 pmol each) and 1 μl of template cDNA. The cDNA was synthesized from 240 ng of total RNA treated with TURBO™ DNase (Invitrogen) using smART First Strand cDNA Synthesis kit (EurX) according to supplier’s protocol. The qPCR reactions were carried out at the following conditions: 95° for 15 min, followed by 40 cycles of 95° for 15 s, 51.5° for 20 s and 72° for 20 s. The value of crossing threshold cycles (Cq) was determined using the micPCR software v2.8.10. Pfaffl method [88 (link)] was applied to calculate relative expression with respect to that of ACT1 that was used as the normalization reference for target gene expression. Then, the fold difference of the gene expression levels between evolved and starting populations, corrected for efficiency, was calculated.
4
Quantitative Gene Expression Analysis
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Gene expression analysis was performed using RT-qPCR using two steps. First, reverse transcription of RNA was performed using a smART First Strand cDNA Synthesis Kit (0804, EURx, Poland) following the manufacturer's protocol. Second, qPCR was performed with a reaction mixture of 12.5 µL containing 20 ng of cDNA, 1 µM each of forward and reverse primers (Sigma-Aldrich, Germany), and 6.25 µL of SG qPCR Master Mix (2 ×) (0401, EURx, Poland). Two technical replicates of each qPCR reaction were performed in 96 well plates (4TI-0955, AZENTA, Poland). Thermo-cycling protocol involved a pre-incubation step (at 95 °C for 15 min) with 40 cycles of subsequent denaturation (95 °C for 15 s), annealing (58˚C for 30 s), and elongation (72 °C for 30 s) steps using a LightCycler 480 II (Roche-Diagnostics, Switzerland). The average Ct values of the technical replicates were used to calculate the relative gene expression of the selected genes using the ΔΔCt method (Livak and Schmittgen 2001) (link). The details of the genes selected for analysis along with their primer sequences are outlined in Table 2 Table 2 Primers used for the qPCR to determine the relative gene expression in cecal mucosa Full size table
5
Silymarin Regulates SAP4 Expression in C. albicans
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The expression of the SAP4 gene was evaluated using real-time PCR after treatment with silymarin. The cells of C. albicans ATCC 10231 were exposed to silymarin at the concentration of 15 µg/mL (MIC/2) or 1% DMSO (control) during propagation in liquid culture in Spider medium (1% nutrient broth, 1% mannitol, 0.2% K2PO4 (Sigma-Aldrich), pH 7.2) with 10% fetal bovine serum (FBS) (Sigma-Aldrich). After 20-h incubation at 37 °C, the yeasts were collected and total RNA was extracted with the YeaStar RNA kit (Zymo Research, USA), according to the manufacturer’s instructions. cDNA was then synthesized using a Smart First Strand cDNA Synthesis Kit (EurX, Poland) following the manufacturer’s instructions. For PCR detection of transcripts, we used TaqMan gene expression assays (Lot: 170255, designed by manufacturer, Thermo Fisher Scientific, England) and the Probe qPCR Master Mix (EurX, Poland). The cDNA samples were pre-treated at 50 °C for 2 min with uracil-N-glycosylase to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95 °C for 10 min., followed by 40 amplification cycles with denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s using RotorGene-6000 (Corbett). The relative level of expression of the tested gene was calculated with the 2-(ΔΔCt) method using ACT1 as a reference gene [38 (link)].
6
Quantitative gene expression analysis
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First-strand cDNA synthesis was performed with total RNA and random hexamer primers using smART First Strand cDNA Synthesis Kit (Eurx, Poland) according to the manufacturer's instructions. Reference genes were selected in separate qPCR among HPRT1, TFRC, ACTB, TBP, and PPIH genes for rat samples and among HPRT1, ACTB, GUSB, and PPIH genes for mouse samples. In both cases, ACTB showed a stable expression in the examined samples and was chosen as an endogenous positive control.
The expression of TNFα, IL-6, Gadd45a, COL1A1, COL3A1, TGFβ, CYP2E1, PPARα, C-met, and HGF genes (Table 2, Ref. [47] [48] [49] [50] [51] [52] ) was detected using FastStart Essential DNA Green Master (Roche, Switzerland) in Light Cycler 96 (Roche, Switzerland). All samples were tested in triplicate. Oligonucleotide primers used for the reactions were purchased from Sigma Aldrich Company (USA). Each run was completed using melting curve analysis to confirm the specificity of the amplification and the absence of primer dimers. The relative expression of the examined genes was calculated according to the 2 -∆∆Ct method.
The expression of TNFα, IL-6, Gadd45a, COL1A1, COL3A1, TGFβ, CYP2E1, PPARα, C-met, and HGF genes (Table 2, Ref. [47] [48] [49] [50] [51] [52] ) was detected using FastStart Essential DNA Green Master (Roche, Switzerland) in Light Cycler 96 (Roche, Switzerland). All samples were tested in triplicate. Oligonucleotide primers used for the reactions were purchased from Sigma Aldrich Company (USA). Each run was completed using melting curve analysis to confirm the specificity of the amplification and the absence of primer dimers. The relative expression of the examined genes was calculated according to the 2 -∆∆Ct method.
7
Gene Expression Analysis in Cerebral Embolism
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Total RNA extraction was performed from serum of the patients and healthy controls using RNA Extracol (EURx, Poland) following the manufacturer's instructions. The RNA was quantified by FLUOstar Omega Microplate Reader (BMG LABTECH, Germany) using LVis plate. For cDNA synthesis, smART First Strand cDNA Synthesis kit (EURx, Poland) was used according to manufacturer's recommendations. Expression levels of SELP (P-selectin) and RETN (resistin) genes were determined in the serum of CE patients and healthy controls. The primers for SELP and RETN genes were selected according to previous studies (15,16). RT-PCR were performed using SYBR Green Master Mix (A.B.T., Turkey) and primers at 200 nM final concentration with ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific). The relative expression of SELP and RETN genes were determined using comparative CT (∆∆CT) method between CE patients and healthy controls. For normalization, GAPDH gene was used.
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