H9c2 cells were washed with phosphate-buffered saline (PBS) and were lysed using the RLT reagent (Qiagen, Hilden, Germany). Total RNA was isolated with the RNeasy micro kit that included DNase digestion step (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was transcribed with SensiFAST cDNA Synthesis Kit (#BIO-65054, Bioline, London, UK), and real-time quantitative-PCR was performed using the SensiFAST SYBR No-ROX (Bioline) and QuantiTect® primers according to manufacturer’s instructions (QuantiTect®, Qiagen; the sequences are not available due to company’s policy). Results were analyzed using the delta-deltaCT method [46 (link)], with 28SrRNA as a reference gene, and represented as normalized values of normoxic scrambled control treated cells.
Quantitect
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, France, Canada, Netherlands
QuantiTect is a portfolio of real-time PCR kits for gene expression analysis. The kits include reagents and primer assays designed for reliable and sensitive quantification of RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (37 mentions)
Trizol reagent, by Thermo Fisher Scientific (20 mentions)
Trizol, by Thermo Fisher Scientific (14 mentions)
Rneasy kit, by Qiagen (12 mentions)
Lightcycler 480, by Roche (12 mentions)
Rneasy plus mini kit, by Qiagen (11 mentions)
Iscript cdna synthesis kit, by Bio-Rad (11 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (9 mentions)
Steponeplus, by Thermo Fisher Scientific (9 mentions)
Quantitect reverse transcription kit, by Qiagen (8 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (8 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (6 mentions)
Nanodrop, by Thermo Fisher Scientific (5 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (5 mentions)
Nucleospin rna kit, by Macherey-Nagel (5 mentions)
Tri reagent, by Merck Group (5 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Mirneasy mini kit, by Qiagen (5 mentions)
Icycler, by Bio-Rad (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
227 protocols using quantitect
1
Quantitative Analysis of H9c2 Cell RNA
2
RNA Extraction, cDNA Synthesis, and qPCR Analysis
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RNA was isolated from cells using the RNeasy kit (Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. Genomic DNA was removed by on-column DNA digestion with RNase-Free DNase Set (Qiagen, Germantown, MD, USA). RNA quality and concentration was assessed using a spectrophotometer (NanoDrop, Thermo-Fisher Scientific, Waltham, MA, USA) and by electrophoresis on a 2% agarose gel. One microgram of RNA was used to synthesize cDNA using random hexamer priming and SSRT-III reverse transcriptase (Life Technologies, Carlsbad, CA, USA), which was followed by qPCR using Quantitect (Qiagen, Germantown, MD, USA) primer assays. Data were normalized against Rrn18S gene transcripts (Quantitect, Qiagen, Germantown, MD, USA). Data were derived from at least three independent biological replicates, and are represented as mean ± SEM values. Data were analyzed using the delta-delta Ct method. Statistical analyses were performed using the GraphPad Prism software, version 7.0.(https://www.graphpad.com/scientific-software/prism/ ).
3
Quantifying PLCγ1 mRNA Abundance
4
Total RNA Extraction and qPCR Analysis
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Total RNA was extracted from cells using RNeasy kit (Qiagen) and checked for integrity using agarose gel electrophoresis. One microgram of RNA was used to synthesize cDNA using random hexamer priming and SSRT-III reverse transcriptase (Life Technologies), followed by qPCR using Quantitect (Qiagen) primer assays or primers designed and ordered from IDT (see Supplementary Table 1 ). Data were normalized against Rrn18S gene transcripts (Quantitect, Qiagen). Data were derived from at least three independent biological replicates, and are represented as mean ± SEM values. Data were analyzed using the delta-delta Ct method as described previously [43 (link), 56 (link)]. Statistical analyses were performed using the GraphPad Prism software, version 7.0.
5
Quantitative Gene Expression Analysis of Canine CrCL
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Each intact CrCL was snap frozen and pulverized (BioPulverizer; BioSpec Products Inc) in liquid nitrogen. Each CrCL and construct tissue specimen was then homogenized in a phenol-guanidinium thiocyanate solution (Sigma-Aldrich), and RNA was extracted in accordance with the manufacturer's instructions.
Messenger RNA was reverse transcribed (Quanti-Tect; Qiagen) into cDNA. Canine-specific primers for the genes Col1, collagen type III, biglycan, decorin, tenascin-c, fibronectin, and tenomodulin were designed from published gene sequences in accordance with National Center for Biology Information standards (Appendix). Relative gene expression was determined by use of SYBR green reagents (Quanti-Tect; Qiagen) and a real-time PCR assay system (7900 Real-Time PCR System; SDS version 2.4; Applied Biosystems). Gene expression data from specimens was normalized to the reference gene (Glutaraldehyde phosphate dehydrogenase [GADPH]), and fold change with cycle threshold (Ct; 2 -ΔΔCt ) in gene expression for specimens cultured in ligamentogenic medium relative to specimens cultured in stromal medium was determined as described. 34 Gene expression data for the native CrCLs were likewise normalized to GADPH and reported as the ΔCt.
Messenger RNA was reverse transcribed (Quanti-Tect; Qiagen) into cDNA. Canine-specific primers for the genes Col1, collagen type III, biglycan, decorin, tenascin-c, fibronectin, and tenomodulin were designed from published gene sequences in accordance with National Center for Biology Information standards (Appendix). Relative gene expression was determined by use of SYBR green reagents (Quanti-Tect; Qiagen) and a real-time PCR assay system (7900 Real-Time PCR System; SDS version 2.4; Applied Biosystems). Gene expression data from specimens was normalized to the reference gene (Glutaraldehyde phosphate dehydrogenase [GADPH]), and fold change with cycle threshold (Ct; 2 -ΔΔCt ) in gene expression for specimens cultured in ligamentogenic medium relative to specimens cultured in stromal medium was determined as described. 34 Gene expression data for the native CrCLs were likewise normalized to GADPH and reported as the ΔCt.
6
Quantitative PCR Analysis of Liver Samples
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qPCR was performed as previously reported (7 (link), 12 (link)) with some modifications. Snap-frozen livers or cultured hepatocytes were homogenized in Trizol reagent (Invitrogen, catalog 15596026). RNA isolated according to the manufacturer’s protocol was reverse-transcribed using the Qiagen Quantitect reverse transcriptase kit (Qiagen, catalog 205310). cDNA was subjected to qPCR using the SYBR Green master mix reagent (Applied Biosystems, catalog 4309155). Expression data are depicted as a mean ratio of target gene expression relative to 36B4 (housekeeping gene) expression in each sample, ± SEM. Primer sequences for qPCR are shown in Supplemental Table 1 .
7
LV Gene Expression Quantification by qRT-PCR
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LV gene expression was quantified for 6 animals per group by quantitative RT-PCR as described elsewhere (Champetier et al., 2009 (link)). Pre-optimized primers were from QuantiTect (Qiagen) and IDT (Coralville, Iowa) (Table 1 ) and SsoAdvanced Universal SYBR Green Supermix (Bio Rad, Hercules, CA) was used. We used one pair of non-pre-optimized primers for the enoyl CoA hydratase, short chain 1 gene (Echs1) (5′-GCTTTCAGGGTGTCTTGATTTG-3′ and 5′-GAGCTATGCACTGCAGATAGT-3′; 95 bp transcript). We tested three different genes as possible housekeeping gene as control for this study. Cyclophilin A gene was chosen since it had the one most stable expression among the different groups.
8
RNA Extraction and RT-qPCR for Circadian Genes
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The dissected tissue was then processed for RNA extraction using the ReliaPrep RNA Tissue Miniprep System (Promega, Madison, WI, USA) and the RNA obtained was stored in water at −80°C. Reverse transcription was performed using the High‐Capacity RNA‐to‐cDNA Kit (Applied Biosystems, Foster City, CA, USA), using the same amount of RNA (200 ng) for each sample. Subsequently, real‐time PCR was performed using PowerUp SYBR Green Master Mix (ThermoFisher Scientific, Vilnius, Lithuania) and measured with the StepOnePlus Real‐Time PCR System (Applied Biosystems). Transcripts were amplified using the QuantiTect primer assay (Qiagen, Hilden, Germany) for Hcrtr1, Hcrtr2 and Adcyap genes, with Gapdh as a housekeeping gene. The ΔΔCT method was used for data analysis with Gapdh as a reference gene and ZT0 mean values for relative target gene expression (RQ = 1).
9
Evaluating Goose Tissue-Specific TLR2-1 Expression
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Total RNA was extracted from geese tissues (from six 10-week-old Magang geese) using RNAiso Plus (TaKaRa) and reverse-transcribed using QUANTITECT® (Qiagen, Germany). qPCR was performed using a SYBR® green polymerase mix (TaKaRa). The primers (Table 3 ) used for qRT-PCR were designed by using the primer3 software (http://bioinfo.ut.ee/primer3/ ). To validate qRT-PCR purified products were cloned into pMD19-T (Takara) and sequenced to verify the correct target amplification. PCR products were amplified using a LightCycler 480 (95°C, 15 s; 61°C, 15 s; 72°C, 15 s). The data are presented as mean +/− SD, and were normalized using the β-actin as a house-keeping gene and calculated by the delta Ct method. We compared the gTLR2-1 expression in the indicated tissues with that in the lowest expression tissue. Tissues used for the study included heart, liver, spleen, lung, kidney, jejunum, ileum, cecum, colon, brain, crop, pancreas, proventriculus, gizzard, duodenum, bursa, breast muscle and skin.
10
Macrophage Phenotyping via qPCR
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For further macrophage phenotyping, qPCR analyses were carried out. Total RNA from macrophage cultures was isolated using TriFast (peqlab, Darmstadt, Germany) and phenol-chloroform extraction, and 0.5 μg RNA was transcribed into cDNA using a reverse transcription kit by Qiagen (QuantiTect®, Hilden, Germany) by following the manufacturer's recommendations. qPCR then was conducted with a Thermo Scientific (Waltham, MA, USA) SYBR Green kit. Normalization of qPCR measurements was performed using two housekeepers (Table 2 ). Bio-Rad primers (Table 2 , Bio-Rad Laboratories, Inc., Hercules, CA, USA) were used according to the manufacturer's instructions.
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