Total RNA was extracted using the Qiagen (GmbH, Hilden, Germany) RNAEasy Mini kit. The quantitative real-time RT-PCR was carried out for the analysis of host gene expression in muscles using gene-specific primers from Quanti Tect primer assay kit and Quanti Fast one-step RT-PCR kit (Qiagen, Germany). The thermal profile consists of 10 min of reverse transcription at 50°C one cycle and 5 min of polymerase activation at 95°C, followed by 40 cycles of PCR at 95°C for 10 s, 60°C for 30 s for combined annealing/extension. Following amplification, a melting curve analysis was performed to verify the authenticity of the amplified product by its specific melting temperature (Tm) with the melting curve analysis software of the Mx3005p. The threshold cycle (Ct) of gene of interest (GOI) and housekeeping gene (HKG) and the difference between their Ct values (Ct) were determined. Relative changes of gene expression were calculated using delta delta Ct method [31] (link) and the data were represented as fold up-regulation/down-regulation compared to the mock-infected group.
Quantitect primer assay kit
Manufactured by Qiagen
Sourced in Germany
The QuantiTect Primer Assay kit is a laboratory equipment product manufactured by Qiagen. The kit contains ready-to-use primer assays designed for real-time quantitative PCR (qPCR) analysis. The core function of the product is to facilitate gene expression analysis by providing pre-designed, validated primer pairs for specific target genes.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rt2 sybr green qpcr master mix kit, by Qiagen (2 mentions)
M mlv reverse transcriptase, by Merck Group (2 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Quanti fast one step rt pcr kit, by Qiagen (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
E method, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Sybr green pcr master kit, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
13 protocols using quantitect primer assay kit
1
Quantitative RT-PCR for Host Gene Expression
2
Quantitative Real-time RT-PCR Protocol
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The quantitative real-time RT-PCR was carried out for the selected genes using gene specific primers from Quantitect primer assay kit (Qiagen, Germany). QuantiFast one step RT-PCR kit was used for real time PCR and RNA polymerase-II (RP-II) was used as an endogenous reference gene. Briefly, the reaction mixture consisted of 12.5 μl of 2 × QuantiFast SYBR Green RT-PCR Master Mix, 2.5 μl 10 × Quantitect primer mix, 0.25 μl of Quantifast RT Mix, 100 ng (2 μl) of template RNA and 8.75 μl nuclease free water in 25 μl reaction volume. The Roche Light cycler-480 system was used to monitor the SYBR Green signal at the end of each extension period for 40 cycles. The thermal profile consisted of 10 min of reverse transcription at 50°C for one cycle and 5 min of polymerase activation at 95°C, followed by 40 cycles of PCR at 95°C for 10 s, 60°C for 30 s for combined annealing/extension. The relative quantification levels in expression were determined using the 2nd derivative maximum analysis with the determination of the crossing points for each transcript. Crossing point values for each target gene were normalized to the respective crossing point values for the reference gene RP-II. Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method [44 ].
3
Quantitative PCR analysis of PRKD1 expression
4
RNA Extraction, cDNA Synthesis, and qPCR Analysis
5
Quantitative RT-PCR Analysis of Mouse NOX4
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Total RNAs were extracted from cells with RNeasy® Mini Kit (Qiagen: Hilden, Germany) and reverse-transcribed by SuperScriptTM II reverse transcriptase with Oligo dT primers (Invitrogen: CA, USA). The resultant cDNAs for mouse NOX4 were quantitatively amplified with real-time RT-PCR using QuantiTect® Primer Assay kit (Cat No; QT00126042 for NOX4, Qiagen, Hilden, Germany) and DyNAmoTM HS SBYR®green qPCR kit (Thermo scientific: MA, USA). Standard curve was obtained by plotting Ct (cycle threshold) values against log cDNA concentrations of five serial dilutions of the target nucleic acid. GAPDH was used as an internal control.
6
Quantitative Analysis of Retinoic Acid Pathway
7
RNA Extraction and Real-time PCR Protocol
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Total RNA was isolated from cells using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA), and 1–2 μg RNA was used to generate cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR was performed on an Mx300P QPCR system (Stratagene) using the RT2 SYBR Green qPCR Master Mix Kit (Qiagen) and QuantiTect Primer Assay Kit (Qiagen). All RT-qPCR reactions were performed in triplicate and normalized to the β-actin housekeeping gene. The primer sequences and PCR conditions are available upon request. Dissociation curves were run to verify the singularity of the PCR product. The data were analyzed using the comparative threshold cycle (Ct) method.
8
Gene Expression Analysis of Neuroinflammation
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Total RNAs were isolated from frozen brain samples homogenized in Trizol (Life Technologies) and chloroform using glass beads and the RNeasy Lipid Tissue kit (Qiagen Corp.). qRT-PCR was performed using the Superscript II reverse transcriptase kit (Invitrogen) and SYBR green PCR master kit (Applied Biosystems) and the following pairs of primers for TNF-α: 5’-GACCCTCACACTCAGATCATCTTCT-3’ and 5’-CCTCCACTTGGTGGTTTGCT-3’; IL-1b: 5’- CTGGTGTGTGCAGTTCCCATTA-3’ and 5’-CCGACAGCACGAGGCTTT-3’; I L-1Ra: 5’-CTTTACCTTCATCCGCTCTGAGA-3’ and 5’-TCTAGTGTTGTGCAGAGGAACCA-3’; vimentin:: 5’-CGGAAAGTGGAATCCTTGCA-3’ and 5’-CACATCGATCTGGACATGCTGT-3’; GFAP: 5’-GGGGCAAAAGCACCAAAGAAG-3’ and 5’-GGGACAACTTGTATTGTGAGCC-3’. The primers used for VCAM, ICAM and P-Selectin were from the QuantiTect primer assay kit (Qiagen). Results are expressed following normalization using 18S RNA.
9
Cardiac gene expression analysis
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Hearts/ventricles were harvested from anesthetized mice and processed for real-time PCR (RT-PCR) analysis as previously described [22 (link)]. In addition to the primer pairs that have been previously described [22 (link), 23 (link)], the following were used: Hspa5 (GRP78/BiP), PrimerBank ID number 31981722a1; Hsp90b1 (GRP94), PrimerBank ID number 6755863a1; Casp12 (Caspase 12), PrimerBank ID number 31981868a1; Ddit3 (CHOP), PrimerBank ID number 31982415a1; Eif2ak3 (PERK), PrimerBank ID number 6857781a1; Acox1, PrimerBank ID number 26333821a1; Fabp3, PrimerBank ID number 6753810a1; Orai1, PrimerBank ID number 93277106b1; Stim1, PrimerBank ID number 31981983a2; Hax1, PrimerBank ID number 6754160a1; Rcan2, PrimerBank ID number 46560586c1; and Pparγ, PrimerBank ID number 187960104c1; primers for Pln (phospholamban) were adapted from PrimerBank ID number 213512815c1 (forward primer: 5′ AAGTGCAATACCTCACTCG 3′, reverse primer: 5′ GATCAGCAGCAGACATATC 3′). mRNA levels for Atp2b1 (QT01072106) and Atp2b4 (QT01076271) were determined using QuantiTect Primer Assay Kits (Qiagen).
10
RNA Extraction and qRT-PCR Analysis
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Total RNA extraction was carried out using TRIzol reagent (Life Technologies) following the manufacturer’s instructions. One microgram of total RNA was reverse transcribed using random nonamer primers and MMLV reverse transcriptase (Sigma Aldrich, Inc, USA) according to the manufacturer’s instructions.
qRT-PCR was carried out with 50 ng of cDNA template, QuantiTect primer assay kits (Qiagen, Hilden, Germany) and SYBR green real-time PCR reagent on a CFX96 Touch real-time system in a 96-well plate. The cycler conditions were as follows: (i) 95 °C for 15 min, 1 cycle and (ii) 3-step cycling: 94 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, 40 cycles. Data were normalized to HPRT housekeeping gene.
qRT-PCR was carried out with 50 ng of cDNA template, QuantiTect primer assay kits (Qiagen, Hilden, Germany) and SYBR green real-time PCR reagent on a CFX96 Touch real-time system in a 96-well plate. The cycler conditions were as follows: (i) 95 °C for 15 min, 1 cycle and (ii) 3-step cycling: 94 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, 40 cycles. Data were normalized to HPRT housekeeping gene.
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