Total RNA was extracted with Trizol and 500 μg of RNA was reverse transcribed to complementary DNA (cDNA) using the first-stranded cDNA synthesis kit (Invitrogen). Specific genes were amplified and quantitated by real-time PCR using primers described in Supplementary Table 3 . Real-time PCR was performed using the StepOnePlus real-time detection system and the Power SYBR Green PCR Master Mix (Applied Biosystems). Fluorescent measurements were recorded during each annealing step. The PCR quality and specificity were verified by melting curve analysis and gel electrophoresis. Relative expression was determined by normalizing to the GAPDH endogenous control. These experiments were carried out in duplicate and independently repeated three times.
Steponeplus real time detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The StepOnePlus real-time detection system is a laboratory instrument designed for quantitative real-time PCR (qPCR) analysis. The system enables the detection and quantification of nucleic acid sequences in real-time, providing researchers with accurate and reliable data for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
First stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taqman primer probe set, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Iq supermix, by Bio-Rad (1 mentions)
Icycler real time detection system, by Bio-Rad (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Rneasy minelute cleanup, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
7 protocols using steponeplus real time detection system
1
Real-Time PCR Analysis of Gene Expression
2
Quantitative PCR Analysis of Gene Expression
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Each biological sample used in the quantitative PCR experiments corresponded to a pool of CC from three to four mice. Total RNA was extracted from frozen CC of control and regularly treated mice using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. RNA concentration and purity were determined by a Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA), and RNA integrity was checked by electrophoresis in 1.2% non-denaturing agarose gels.
Total RNA (1.5 μg) was checked for DNA contamination by conventional PCR and then retro-transcribed with Superscript III (200 U/μL; Invitrogen, Carlsbad, CA, USA) using 100 pmol of random hexamers for priming. Subsequent amplifications were performed in a StepOne Plus real-time detection system (Applied Biosystems) using the SYBR Green PCR master mix (Applied Biosystems). The following genes were analyzed: Rock1 (which encodes ROCK1), Rock2 (ROCK2), Nos1 (nitrergic nitric oxide synthase), Nos3 (endothelial nitric oxide synthase), Gucy1a3 (soluble guanylate cyclase), Pde5a (phosphodiesterase type 5), and Prkg1 (protein kinase G). The constitutive HPRT gene was used as the endogenous standard. Primers used in this work are listed inTable S1 .
Total RNA (1.5 μg) was checked for DNA contamination by conventional PCR and then retro-transcribed with Superscript III (200 U/μL; Invitrogen, Carlsbad, CA, USA) using 100 pmol of random hexamers for priming. Subsequent amplifications were performed in a StepOne Plus real-time detection system (Applied Biosystems) using the SYBR Green PCR master mix (Applied Biosystems). The following genes were analyzed: Rock1 (which encodes ROCK1), Rock2 (ROCK2), Nos1 (nitrergic nitric oxide synthase), Nos3 (endothelial nitric oxide synthase), Gucy1a3 (soluble guanylate cyclase), Pde5a (phosphodiesterase type 5), and Prkg1 (protein kinase G). The constitutive HPRT gene was used as the endogenous standard. Primers used in this work are listed in
3
Quantitative Gene Expression Analysis
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For quantitative gene expression analysis, 40 cycles of real-time PCR were performed on the StepOnePlus real-time detection system (Applied Biosystems). Every PCR reaction was carried out in duplicates with 2.5 ng of cDNA in a final volume of 12.5 μl Power SYBR® Green PCR Master Mix (Applied Biosystem). StepOneTM Software v2.2 was used for data analysis. Ppia was used as housekeeping gene to normalize target gene expression. We used the following primer sequences for detection of Pdpn transcripts (41 (link)): FW 5′-agagaacacgagagtacaacc-3′; RV 5′-caacaatgaagatccctccgac-3′, Arg1 (42 (link)): FW 5′-TTGGGTGGATGCTCACACTG-3′; RV 5′-TTGCCCATGCAGATTCCC-3′; Ppia: FW 5′-AATTCATGTGCCAGGGTGGTG-3′; RV 5′-TGCCTTCTTTCACCTTCCCAA-3′. Additional primer sequences are given in Table S1 .
4
Quantitative Real-Time PCR Analysis
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For quantitative gene expression analysis, 40 cycles of real-time PCR was performed on the StepOnePlus real-time detection system (Applied Biosystems). Every PCR reaction was carried out in duplicates with 2.5 ng of cDNA in a final volume of 12.5 μL Power SYBR Green PCR Master Mix (Applied Biosystem). StepOne Software v2.2 was used for data analysis. GAPDH protein were used as housekeeping gene to normalize target gene expression. Primer sequences are given in Supplementary Table 1
5
Quantitative PCR Analysis of Gene Expression
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First-strand cDNA was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative PCR reactions included cDNA diluted four-fold, gene-specific TaqMan® primer probe sets (Applied Biosystems®, Foster City, CA), and iQ™ Supermix (Bio-Rad, Hercules, CA). Cycling was performed in triplicate using a StepOnePlus™ Real-Time detection system (Applied Biosystems®, Foster City, CA). Gene-specific amplification was normalized to GAPDH as an internal reference gene and expressed as log2 relative gene expression compared to control spleen using the formula 2-ΔΔCt.
6
RNA Extraction and qPCR Analysis
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Total RNA was extracted from 1 × 105 cells, and cDNA was synthesized with the AMV-reverse transcriptase and random hexamer primers (Roche Diagnostic Corp., Indianapolis, USA). Reverse transcription was performed using a standard procedure (Super Script First-Strand Synthesis System; Invitrogen) using 1 μg of total RNA. QPCR was performed using the iQ SYBR Green Supermix on the iCycler Real-Time Detection system (Bio-Rad, Hercules, CA, USA) on an StepOne Plus Real-Time Detection system (Applied Biosystems, USA) (27 (link)). The gene expression was normalized to GAPDH and the relative amount of mRNA was calculated using the 2−ΔΔCt method. Primer sequences were listed in Table 2
7
Optimized qPCR Workflow for RNA Analysis
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A portion of RNA from each sample was treated with DNase I (Life Technologies), cleaned and concentrated using RNeasy MinElute Cleanup (Qiagen), and 1 µg of cleaned RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Life Technologies) as previously described [ 23] .
Real-time qPCR analyses were conducted on StepOne-Plus real-time detection system (Applied Biosystems) using SYBR green chemistry. Each PCR reaction consisted of 2X SYBR mastermix (Life Technologies), forward and reverse primers (500 nM each; Table 3), and 1 μL cDNA template (diluted 1:5 in nuclease-free water) to a nal volume of 15 μL. Samples were assayed in duplicate with a ve-step, fourfold dilution series of pooled cDNA included in each run to calculate ampli cation e ciency, linearity, and provide inter-run calibration. No-template controls as well as no-reverse transcriptase controls were also included on each run. Cycling conditions consisted of an initial activation of DNA polymerase at 95 °C for 10 min, followed by 40 cycles of 5 s at 95 °C, 20 s at 60 °C, and 10 s at 72 °C with orescence measured at the end of each 72°C step. Melt curve analyses were subsequently performed to ensure ampli cation speci city.
Real-time qPCR analyses were conducted on StepOne-Plus real-time detection system (Applied Biosystems) using SYBR green chemistry. Each PCR reaction consisted of 2X SYBR mastermix (Life Technologies), forward and reverse primers (500 nM each; Table 3), and 1 μL cDNA template (diluted 1:5 in nuclease-free water) to a nal volume of 15 μL. Samples were assayed in duplicate with a ve-step, fourfold dilution series of pooled cDNA included in each run to calculate ampli cation e ciency, linearity, and provide inter-run calibration. No-template controls as well as no-reverse transcriptase controls were also included on each run. Cycling conditions consisted of an initial activation of DNA polymerase at 95 °C for 10 min, followed by 40 cycles of 5 s at 95 °C, 20 s at 60 °C, and 10 s at 72 °C with orescence measured at the end of each 72°C step. Melt curve analyses were subsequently performed to ensure ampli cation speci city.
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