The relative gene expression of the COX-2 and TNF-α were determined in the liver and spleen tissues of the different tested groups. Total RNA was extracted using the Purelink™ RNA kit (Thermo Scientific, Waltham, MA, USA), and then converted into cDNA by the Power™ cDNA kit (iNtRON Biotechnology, Seongnam, Kyonggi-do, Republic of Korea) to synthesize the cDNA. Rotor-Gene Q 5plex (Qiagen, Hilden, Germany) was utilized in the qRT-PCR run, and the employed primers are revealed in Table S1 .
Rotor gene q 5plex
Manufactured by Qiagen
Sourced in Germany, United States
The Rotor-Gene Q 5plex is a real-time PCR cycler that enables the simultaneous detection of up to five different targets in a single reaction. It features a compact design and supports multiple sample formats.
Automatically generated - may contain errors
Lab products found in correlation
Purelink rna mini kit, by Thermo Fisher Scientific (6 mentions)
Quantitect sybr green pcr kit, by Qiagen (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Quantinova sybr green rt pcr kit, by Qiagen (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Power cdna synthesis kit, by iNtRON Biotechnology (2 mentions)
Sybr green master mix, by Takara Bio (2 mentions)
Oligo dt 20 primer, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Faststart universal sybr green master, by Merck Group (2 mentions)
Prism 9, by GraphPad (2 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Purelink rna kit, by Thermo Fisher Scientific (1 mentions)
Fastgene ic green 2 qpcr universal mix, by Nippon Genetics (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Kapa sybr fast master mix 2x universal, by Roche (1 mentions)
Snord68 11 sno68, by Qiagen (1 mentions)
37 protocols using rotor gene q 5plex
1
Quantifying COX-2 and TNF-α Expression
2
Quantitative PCR Protocol for Gene Expression
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Quantitative PCR (qPCR) amplification was performed using the SYBR green method in a 20 μL reaction mixture with the FastGene® IC Green 2× qPCR Universal Mix (Nippon Genetics Europe, Düren, Germany) following the manufacturer’s instructions. For each gene a specific primer pair was used (Table 2 ). The qPCR reactions were carried out in a 36 well rotor using Rotor-Gene Q 5plex (Qiagen Inc., Hilden, Germany). The amplification was performed according to the following protocol: 95 °C for 2 min followed by 40 cycles of 95 ℃ for 5 s and annealing temperatures for 30 s. Quantification of gene expression levels was performed using the 2−ΔΔCT method as described in our previous works [27 (link),76 (link),77 (link)]. β-actin was used as an internal control gene, and the median value of the NI group was used as a calibrator.
3
Extraction and Detection of Demodex DNA
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For DNA extraction, samples were centrifuged in a microcentrifuge at 13,000× rpm for 30 min. Once the supernatant was removed, samples were submerged in liquid nitrogen and then disrupted using a homogenizer. Isolation was performed using a commercial kit (Thermo Scientific GeneJET Genomic DNA Purification Kit, cat. no. K0722; Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions with minor modifications (samples were incubated for 24 h with Proteinase K Solution and Digestion Solution).
Real-time PCR [14 (link)] reactions were performed (Rotor-Gene Q 5plex; Qiagen, Hilden, Germany) in a total volume of 20 μl (FastStart Universal SYBR Green Master; Roche Diagnostics GmbH, Mannheim, Germany) using 0.3 µmol/l of each primer and 4 µl of DNA. The following primers were used: D. canis forward, 5′-GAT GAA GCG GCG AGT AAT GTT C-3′, reverse, 5′-GAC TCC ATC TTT TAC GAT GTC TGA TTT-3′. These amplified a 166-bp fragment of the chitin synthase gene. Nuclease-free water was used as a negative control for the PCR. Positive controls were obtained from clinical samples that were previously amplified and sequenced to confirm Demodex. The thermal cycling profile was as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
Real-time PCR [14 (link)] reactions were performed (Rotor-Gene Q 5plex; Qiagen, Hilden, Germany) in a total volume of 20 μl (FastStart Universal SYBR Green Master; Roche Diagnostics GmbH, Mannheim, Germany) using 0.3 µmol/l of each primer and 4 µl of DNA. The following primers were used: D. canis forward, 5′-GAT GAA GCG GCG AGT AAT GTT C-3′, reverse, 5′-GAC TCC ATC TTT TAC GAT GTC TGA TTT-3′. These amplified a 166-bp fragment of the chitin synthase gene. Nuclease-free water was used as a negative control for the PCR. Positive controls were obtained from clinical samples that were previously amplified and sequenced to confirm Demodex. The thermal cycling profile was as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
4
Quantitative RT-PCR Analysis of T. pretiosum
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T. pretiosum cDNA templates were prepared from total RNA samples using the SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. Quantitative Real-Time PCR (qRT-PCR) was performed using gene-specific primers (Supplementary Table S1 ), which were designed using the Primer3web (version 4.1.0) software (http://bioinfo.ut.ee/primer3/ , accessed on 10 August 2019). The reference housekeeping gene, glyceraldehyde-phosphate dehydrogenase (GAPDH), was selected here because it has been shown as one of the best candidate reference genes in qPCR analysis [28 (link)]. A 2-Step qPCR protocol was performed on Rotor-Gene Q-5 Plex (Qiagen, Valencia, CA, USA) using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 65 to 95 °C in increments of 1.0 °C for 5 s [29 (link)]. For each cDNA sample and primer set, reactions were run in triplicate, and average fluorescence Ct values were obtained. Relative expression levels were determined using the comparative 2−ΔΔCt method for relative quantification [30 (link)]. Three biological replicates were performed. Statistical analysis was performed on the expression profiles between male and female adults using the Student’s t-test (SPSS version, IBM, Armonk, NY, USA).
5
Measuring Cytokine Expression in Paw Tissues
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The inflammatory cytokines mRNA expression of IL-1β and IL-6 was studied in the paw tissues [51 (link)]. We used the beta-actin gene [52 (link)] as a housekeeping gene, and the utilized primers are presented in Table S1 . After extraction of the RNA using the Purelink™ RNA Mini Kit (Thermo Scientific, Waltham, MA, USA), RNA was turned into cDNA using the power™ cDNA synthesis kit. qRT-PCR was employed using Rotor-Gene Q 5plex (Qiagen, Hilden, Germany).
6
RT-qPCR Amplification of Gene Expression
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Reverse transcription-quantitative PCR (RT-qPCR) amplification was performed using SYBR green method in a 20 μl reaction mixture with the “KAPA SYBR® FAST Master Mix (2X) Universal”(KAPA Biosystems, USA), in accordance with the manufacturer’s instructions. For RT-qPCR reaction 0.2 μM of each specific primer was used. The primers used for amplification are shown in Table 3 .
The RT-qPCR reactions were carried out in 36-well rotor using “Rotor-Gene Q 5plex”(Qiagen, Valencia, CA). The amplification was performed according to the following protocol: 95°C for 2 min followed by 40 cycles of 95°C for 20 s, 60°C for 30 s and 72°C for 80 s. Quantification of gene expression levels was done using 2−ΔΔCT method [58 (link)]. For normalization of each gene expression β-actin was used as an internal control gene and median value of NI group was used as a calibrator.
The RT-qPCR reactions were carried out in 36-well rotor using “Rotor-Gene Q 5plex”(Qiagen, Valencia, CA). The amplification was performed according to the following protocol: 95°C for 2 min followed by 40 cycles of 95°C for 20 s, 60°C for 30 s and 72°C for 80 s. Quantification of gene expression levels was done using 2−ΔΔCT method [58 (link)]. For normalization of each gene expression β-actin was used as an internal control gene and median value of NI group was used as a calibrator.
7
Bladder Cancer miRNA-21 Expression
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Two tissue samples were obtained from each BC patient: the first was collected from central part of the tumor and the second which served as a control was taken from non-lesion part of the tissue. Tissue samples for miRNA analysis were preserved in RNA later at -80 °C. Total RNA including miRNAs was extracted from bladder tissues using miRNeasy Mini Kit (cat.# 217004, Qiagen, Hilden, Germany), then it reverse transcribed to cDNA in a final volume of 20 μL using the miScript Reverse Transcription kit (cat.# 218161,Qiagen, Hilden, Germany). The qPCR measurements were done in triplicate using miScript SYBR-Green PCR kit (SYBR® Green PCR Kit) (cat.# 218073,Qiagen, Hilden, Germany) and miScript primer assay for miRNA-21 on the Rotor-Gene Q 5-Plex (Qiagen) according to the manufacturer's instructions. The results were normalized to SNORD68_11 (sno68) (Qiagen). The amplification profile was denatured at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min and 70 for 30 °C s. The expression level of miRNA gene was represented by fold change, which was calculated using the equation 2−ΔΔCT (Livak and Schmittgen 2001 (link)).
8
Quantification of GOX Gene Expression in Honey Bees
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From pooled samples containing 50 bees per colony which were kept in DNA/RNA Shield (Zymo Research), the total RNA was isolated using Quick-RNA™ MiniPrep (Zymo Research, Irvine, CA, USA), in accordance with the manufacturer’s instructions. After conversion into cDNA using the FastGene 55-Scriptase cDNA Synthesis set (Nippon Genetics), in compliance with the manufacturer’s instructions, real-time PCR amplification was done using SYBR green method with the KAPA SYBR® FAST qPCR Kit (KAPA Biosystems, Wilmington, MA, USA) in accordance with the manufacturer’s instructions: reaction mixture (20 µL) contained 1× KAPA SYBR FAST qPCR Master Mix (2×) Universal, 200 nM of each primer, 1 μL (5 ng) of cDNA. Primers by Yang and Cox-Foster [42 (link)] are given in Table 1 . The qPCR reactions were carried out in triplicate in “Rotor-Gene Q 5plex” (Qiagen) using following thermal protocol: 2 min at 95 °C, 40 cycles of amplification with 20 s at 95 °C, 30 s at 60 °C and 80 s at 72 °C. The relative quantification was done with the 2−ΔCt method [43 (link)] using β-actin as an internal control gene, for the normalisation of GOX gene expression.
9
Quantifying Immune Gene Expression
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We used qPCR to analyze the expression of Tppgrp-lc and Tptoll receptors and Tprelish as well as of prolixicin, defensin B, and lysozyme B in individual cDNA samples of fat body tissue after the challenge with M. luteus and E. coli. Each reaction was performed in a final volume of 10 µl, containing 1 µl of cDNA (1:20), 1.5 pmol of each oligonucleotide, and 5 µl of SYBR Green 2X Mix (NZY qPCR Green Master Mix, nzytech, Lisbon, Portugal). qPCR was performed on a Rotor-Gene Q 5plex (Qiagen, Hilden, Germany). The amplification efficiency for each transcript was analyzed (by serial dilutions of the cDNA sample) using the standard curve method, with the formula E = 10(−1/slope) − 1 (r = 0.94). The qPCR conditions used were as follows: 95 °C for 3 min, 40 cycles of 95 °C for 15 s and 61 °C for 1 min, followed by melt curve analysis to confirm the specificity of the reaction and 1.2% agarose gel electrophoresis to determine the molecular weight. Controls without templates were included with each primer set, to verify the absence of exogenous DNA and oligonucleotide dimers.
10
Relative Expression of CCR2 and CMKLR1
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Relative expression of CCR2 and CMKLR1 analysis:
To ensure data accuracy, experiments were done in duplicate, and blank and internal controls were included in all reactions. A threshold cycle (CT) value was determined from each amplification plot. Data was collected with applications of Rotor-Gene Q 5-Plex detector software (QIAGEN). The increase of relative expression in the target genes was calculated using the comparative CT method with 2−ΔΔCT equation [36 (link)].
To ensure data accuracy, experiments were done in duplicate, and blank and internal controls were included in all reactions. A threshold cycle (CT) value was determined from each amplification plot. Data was collected with applications of Rotor-Gene Q 5-Plex detector software (QIAGEN). The increase of relative expression in the target genes was calculated using the comparative CT method with 2−ΔΔCT equation [36 (link)].
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