The isolated total RNA (1 µg) was reverse-transcribed to produce cDNA using iScript Reverse Transcription Supermix kit (Bio-Rad, Hercules, CA, USA). Commercially available QuantiTect primers (Qiagen, Hilden, Germany) for IL-6, monocyte chemoattractant protein-1 (MCP-1), and IL-1β were used and primer sequences for a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), aggrecan (ACAN), type I collagen (COL1A1), type II collagen (COL2A1), heparanase (HPSE), matrix metalloproteinase-3 (MMP3), matrix metalloproteinase-9 (MMP9), and tissue inhibitor of metalloproteinase 3 (TIMP3) genes obtained from existing literature (Table 1 ). Real-time PCR was performed using SYBR Green supermix (Bio-Rad). Relative gene expression was expressed as fold change in gene expression between mock-infected and RRV-infected samples, with threshold cycle (CT) values normalised using the HPRT1 housekeeping gene (QuantiTect primer, Qiagen).
Quantitect primer
Manufactured by Qiagen
Sourced in United States, Germany, United Kingdom, Australia, Switzerland, Netherlands, France
QuantiTect primers are a range of pre-designed, gene-specific primer sets for real-time PCR analysis. They are optimized for use with QuantiTect and QuantiFast qPCR kits to provide reliable and sensitive detection of target genes.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (43 mentions)
Quantitect reverse transcription kit, by Qiagen (22 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (21 mentions)
Rneasy kit, by Qiagen (18 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (17 mentions)
Trizol reagent, by Thermo Fisher Scientific (17 mentions)
Trizol, by Thermo Fisher Scientific (13 mentions)
Cfx384 real time pcr detection system, by Bio-Rad (12 mentions)
Quantitect sybr green pcr kit, by Qiagen (12 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (11 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (10 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (9 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (8 mentions)
Iscript cdna synthesis kit, by Bio-Rad (8 mentions)
Quantifast sybr green kit, by Qiagen (7 mentions)
Tetro cdna synthesis kit, by Meridian Bioscience (7 mentions)
Lightcycler 480, by Roche (7 mentions)
Oligo dt primer, by Thermo Fisher Scientific (7 mentions)
Quantifast sybr green pcr kit, by Qiagen (6 mentions)
Rneasy plus mini kit, by Qiagen (6 mentions)
247 protocols using quantitect primer
1
RNA Expression Analysis of Inflammatory and ECM Genes
2
qPCR Analysis of ACKR3 and CGRP Receptor Components
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Total cellular RNA was extracted from confluent plates of human LECs or HEK293 control (HEK293-CTRL) cells and HEK293 cell stably transfected with ACKR3 (HEK293-ACKR3) using TRIZOL reagent (Life Technologies, cat. no.15596026). cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific, cat. no. 10400745). Quantitative PCR was performed on reversely transcribed RNA using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, cat. no. A25776). Gene expression analysis was performed on a QuantStudio 7 Flex system (Applied Biosciences) using the following primer pairs:
Ackr3 (RV:5’-GTA GAG CAG GAC GCT TTT GTT-3’ ; FW: 5’-TCT GCA TCT CTT CGA CTA CTC- 3’ ); Calcrl (RV: 5’-CAT CAA TGG TGT GCT GGA AC-3’ ; FW: 5’-CAC TAT GCC TGA TGT GAC GC-3’ ); Ramp2 (RV: 5’-GTT GGC AAA GTG GAT CTG GT- 3’ ; FW: 5’-GCC ATG ATT AGC AGG CCT TA-3’ ); Ramp3 (RV: 5’-CTC ATC CCG CTG ATC GTT AT- 3’ ; FW: 5’AAC TTT CTT CCA GCT TGC CA- 3’ ); β-actin (ACTB) (QuantiTect primer, Qiagen, cat no. QT01680476). Cxcr4 (QuantiTect primer, Qiagen, cat no. QT00223188).
Ackr3 (RV:
3
Achilles Tendon Injury and Repair Dynamics
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Animals were euthanized with anesthetic overdose. Achilles tendons were isolated from surrounding tissue and dissected from both sides of hind limbs. The proximal ends of the Achilles tendons were disconnected at the end of the gastrocnemius, plantaris, and soleus, and the distal ends at the calcaneus. Left leg tendons of eight control rats and six burn rats for each time point (1, 3, 7, and 14 days) were stored in RNAlater (Qiagen), whereas right leg tendons were snap frozen for protein extraction. Samples were stored in −80°C for further analysis. Tendon RNA was obtained with RNAeasy Universal kit (Qiagen). cDNA conversion was done with iScript kit (Bio-Rad), and qPCR with SsoFast Eva Green kit (Bio-Rad). Primers for IL-6, TNF, IL-1β, col1a1, col3a1, MMP9, MMP13, and TGFβ1 were purchased from QuantiTect Primers (Qiagen). Gene expression was calculated using the ΔΔCt method with 18 s as housekeeping.
For protein extraction, tendon tissue was homogenized in RIPA buffer (Invitrogen) with proteinase inhibitor cocktail (Sigma). Protein quantification was performed with BCA assay (Pierce); 10 μg of total protein was used for Western blot. Antibodies included anti-collagen I (Abcam), anti-collagen III (Abcam), goat anti-mouse HRP (Pierce), and goat anti-rabbit HRP (Pierce), which were prepared in 2% BSA. Blots were developed with ECL (Pierce) using a CCD camera system.
For protein extraction, tendon tissue was homogenized in RIPA buffer (Invitrogen) with proteinase inhibitor cocktail (Sigma). Protein quantification was performed with BCA assay (Pierce); 10 μg of total protein was used for Western blot. Antibodies included anti-collagen I (Abcam), anti-collagen III (Abcam), goat anti-mouse HRP (Pierce), and goat anti-rabbit HRP (Pierce), which were prepared in 2% BSA. Blots were developed with ECL (Pierce) using a CCD camera system.
4
Quantitative RT-PCR Analysis of iNOS and Cytokines
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rA2 cells were washed once with phosphate buffered saline (PBS), macrophages were scraped off the surface, packed by centrifugation and washed with PBS. Cells were lyzed with RLT reagent (Qiagen, Hilden, Germany), and total RNA was isolated using the RNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA (0.1 µg) was transcribed with Superscript II reverse transcriptase (Invitrogen, Life Technologies, Langenselbold, Germany) using random hexamere primers (Roche, Mannheim, Germany). Real time quantitative PCR was performed in the LightCycler® (Model 480, Roche, Mannheim, Germany). The ABsolute QPCR SYBRgreen mix (Thermo Scientific, Schwerte, Germany) was used with QuantiTect®-primers (QuantiTect®, Qiagen, Hilden, Germany) for the detection of iNOS and cytokines; 28S-rRNA was used as a housekeeping gene. The 2−△△Ct method was used to analyze the relative mRNA abundance [59 (link)].
5
Activation and Characterization of MSCs
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Activation of MSCs was achieved as described previously 4 . Briefly, maMSCs were seeded into Nunclon 35 mm Petri dishes in DMEM (plus supplements, as above) and allowed to adhere for 24 hours. The medium was replaced by DMEM supplemented with interferon‐ and tumor necrosis factor‐α (TNF‐α; Peprotech, London, United Kingdom; 20 ng/ml each), and the maMSCs were incubated for further 8 hours. MSCs were washed with PBS, trypsinized, and pelleted by centrifugation (1000g, 3 minutes), and mRNA was extracted using a commercially available kit (RNeasy mini kit, QIAGEN, United Kingdom) The expression of mRNAs for chemokine (C‐X‐C motif) ligand 9 (CXCL9) and nitric oxide synthase (NOS2) was quantified against glyceraldehyde‐3‐phosphate dehydrogenase mRNA by quantitative real‐time polymerase chain reaction using QIAGEN QuantiTect primers (QT0010124; QT00100275) as described previously 20 .
6
Quantitative RT-PCR Analysis of Zebrafish Retina
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Quantitative RT-PCR was performed with total RNA extracted from dissected fixed retinae at 28 hpf and 3 dpf. Briefly, embryos were fixed in 4% paraformaldehyde overnight and transferred to RNAse-free PBS, after two washes for 5 min each. Embryo tails were genotyped using KASP assays (Kettleborough et al., 2013 (link)). Heads were pooled by genotype and retinae dissected manually using insect pins. RNA was extracted using RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion) from 40 retinas for each condition (wild-type sibling versus mutant at each time point). RNA quality control was performed with the Experion LabChip (Bio-Rad). cDNA was synthesised and amplified with the Transplex Whole Transcriptome Amplification Kit (Sigma), and quantified using a Nanodrop 2000c. Quantitect primers (Qiagen) were used to amplify col15a1b (QT02215941), ccnd1 (QT02178519), atoh7 (QT02188459), nr2f5 (QT02125424), aldh1a3 (QT02111613), crx1 (QT02229584), cdkn1c (QT02052253) and β-actin (QT02174907). Real-time PCR was performed on a BioRad iCycler using GoTaq qPCR Master Mix (Promega). Fold change in RNA levels was calculated using the ΔΔCt method, and expression normalised to β-actin levels (Livak and Schmittgen, 2001 (link)).
7
Quantitative Real-Time PCR Analysis
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cDNA was generated from 500ng of total RNA using High Capacity RNA-to-cDNA kit (Life Technologies) according to the manufacturer’s instruction. qRT-PCR assays were performed using QuantiTect SYBR Green PCR kit and the following commercially available QuantiTect primers: (Qiagen, Crawley, UK) for COX6C (Assay ID QT00221137), NDUFB2 (Assay ID QT00050904), and LAT (Assay ID QT00232127). Differences in expression were determined by the relative quantification method; the cycle threshold (CT) values of the target genes were first normalised to the CT values of endogenous control large ribosomal protein P0 (RPLPO, Assay ID QT00075012).
8
Cytokine and Cadmium Exposure on RA Synoviocytes
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RA synoviocytes were plated at a density of 2,5x105 cells/cm2 in 12-well plates and were then exposed or not to cytokines overnight followed or not by Cd exposure for 6 hours. After 6 hours of treatment, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, DE) and quantified with the Quant-it kit assay (Invitrogen by Thermo Fisher Scientific, Grand Island, NY, USA) following manufacturer’s instructions. cDNA was synthesized using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s instructions. SYBR green-based real-time qRT-PCRs were performed on the CFX96 Real-Time PCR Detection System (BioRad, Hercules, CA, USA) using the QuantiFast SYBR green kit and QuantiTect primers (Qiagen). Cycle threshold values were normalized with respect to the endogenous control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative expression of the genes in treated cells versus control cells was determined using the comparative threshold cycle method as described by the manufacturer.
9
Axonal RNA Extraction and qRT-PCR Analysis
10
Quantitative PCR of Pancreatic Cells
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