Total RNA was isolated from RAW264.7 macrophages using RNAiso Plus (TaKaRa) [33 (link), 34 (link)]. RNA was converted into cDNA using PrimeScript RT Mix (TaKaRa). Afterward, qRT-PCR experiments were conducted in a LightCycler PCR system (Roche) with UNICONTM SYBR Green (11198ES08, Yeasen). The sequences of all the primers used are shown in Table 1 .
Light cycler pcr system
Manufactured by Roche
Sourced in United States
The LightCycler PCR system is a compact, fully integrated real-time PCR instrument designed for fast, efficient, and precise DNA amplification and analysis. It features a high-intensity blue LED as the light source and utilizes capillary sample containers for rapid thermal cycling. The system enables sensitive and quantitative detection of target DNA sequences in a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (4 mentions)
Sybr green realtime pcr master mix, by Toyobo (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Steponeplus system, by Thermo Fisher Scientific (2 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Primescript rt mix, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Tri reagent rna dna protein isolation reagent, by Molecular Research Center (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Upl probe, by Roche (1 mentions)
Complete protease inhibitor with edta, by Roche (1 mentions)
Sybr green reagent, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
16 protocols using light cycler pcr system
1
RNA Isolation and qRT-PCR in RAW264.7 Macrophages
2
Quantifying MCP-1 and CX3CL1 Gene Expression
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The gene expression levels of MCP-1 and CX3CL1 were assessed by real-time PCR. Total RNA was isolated using the TRI reagent (RNA/DNA/protein isolation reagent, Molecular Research Center, Cincinnati, OH, USA) and reverse-transcribed into cDNA using a random hexamer and M-MLV reverse transcriptase (Promega, Madison, WI, USA) at 23°C for 10 minutes, 42°C for 60 minutes, and 95°C for 5 minutes. The cDNA was incubated with SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) containing 10 pg/mL of forward and reverse primers, and amplified using the Light Cycler PCR system (Roche Applied Sciences, Indianapolis, IN, USA). RT-PCR was performed using the following rat-specific primers for MCP-1: 5’-ACGTGCTGTCTCAGCCAGAT-3’ (forward) and 5’-GTTCTCCAGCCGACTCAT TG-3’ (reverse), CX3CL1: 5’-CACAAGATGACCTCGCCAAT-3’ (forward) and 5’-GCTGTCTCGTCTCCAGGATG-3’ (reverse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5’-GCTCTCTGCTCCTCCCTGTT-3’ (forward) and 5’-CACACCGACCTTCACCATCT-3’ (reverse). For PCR amplification, the cDNA was amplified by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds. During the first cycle, the 95°C step was extended to 3 minutes. GAPDH was amplified in the same reaction as the reference gene.
3
Quantifying AMCase and hrGFP Expression
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The expressions of AMCase and humanized Renilla green fluorescent protein (hrGFP) were analyzed using real-time PCR with a LightCycler PCR system (Roche, Indianapolis, IN). AMCase was amplified using the following primers: forward, 5′-TGGACACACCTTCATCCTGA-3′; and reverse, 5′-AACA AGCCCTGCTTGACAAT-3′. After initial denaturation at 95 °C for 10 min, 45 cycles were performed at 95 °C for 10 s, 50 °C for 10 s, and 72 °C for 10 s. The reaction parameters for hrGFP amplification (forward primer, 5′-ATGGTGAGCAAGCAGATCCTG-3′; reverse, 5′-GGTGCGCTCGTACACGAAGCC-3′) were as follows: initial denaturation at 95 °C for 10 min, followed by 35 cycles at 95 °C for 10 s, 50 °C for 10 s, and 72 °C for 10 s. Actin as a housekeeping gene used the same protocol as hrGFP, but the primers were as followed: forward primer, 5′-GAAACTACATTCAAT TCCATC-3′; reverse primer, 5′-CTAGAAGCACTTGCGGT GCAC-3′.
4
Real-Time qPCR for Rat IL-6 Gene Expression
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Total RNA was isolated using TRI reagent (Molecular Research Center, Cincinnati, OH, USA) and reverse-transcribed into cDNA using a random hexamer and M-MLV reverse transcriptase (Promega, Madison, WI, USA) with conditions at 23 °C for 10 min, 37 °C for 60 min, and 95 °C for 5 min. The cDNA was incubated with SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) that contained 10 pg/mL of forward and reverse primers, and amplified using a Light Cycler PCR system (Roche Applied Sciences, Indianapolis, IN, USA). Real-time PCR was conducted with the following rat-specific primers for IL-6 and β-actin. The IL-6 (accession number NM_012589) primers 5′-GAGAGGAGACTTCACAGAGGATACCA-3′ (forward primer) and 5′-CCACAGTGAGGAATGTCCACA-3′ (reverse primer) were used to generate a 590 bp PCR product. For β-actin, the forward primer used is 5′-ACCAACTGGGACATGGAG-3′ and the reverse primer used is 5′-GTCACGATCTTCATGAGGTAGTC-3′, giving an 890 bp PCR product. For PCR amplification, the cDNA was amplified by 40 repeat denaturation cycles at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 45 s. During the first cycle, the 95 °C step was extended to 3 min. The β-actin gene was amplified in the same reaction as a reference gene.
5
Protein and RNA Expression Analysis
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Cells or tissues were harvested in RIPA buffer plus 1× cOmplete protease inhibitor with EDTA (Roche Applied Sciences) on ice. Western blotting was performed as described previously (16 (link)). Primary and secondary antibodies are listed in Table S1 . Immunoreactive bands were detected by the Odyssey CLx Imaging System. Band intensities were quantified using ImageJ for further statistical analysis.
Total RNA from cells and animal tissues was isolated by the TRIzol reagent (Thermo Fisher; 15596018) according to the supplier's standard protocol. Reverse transcription was performed as reported previously (16 (link)). Primers and specific probes were synthesized at Eurofins MWG Operon. Primers were designed by the Universal Probe Library (Life Science, Roche Molecular Systems), and the UPL probes were also purchased from Roche. The sequences of each primer are shown inTable S2 . qRT-PCR was performed and read by the LightCycler PCR system (Roche Molecular Systems, Inc).
High binding surface microplates for ELISA were purchased from Corning Company (Product No. 9018), and a direct ELISA assay was established by human BMP6 antibody (Novus Biologicals Company, DY507) and recombinant BMP6 protein (R&D Systems, 507-BP-020). ELISA was performed according to the manufacturer's protocol.
Total RNA from cells and animal tissues was isolated by the TRIzol reagent (Thermo Fisher; 15596018) according to the supplier's standard protocol. Reverse transcription was performed as reported previously (16 (link)). Primers and specific probes were synthesized at Eurofins MWG Operon. Primers were designed by the Universal Probe Library (Life Science, Roche Molecular Systems), and the UPL probes were also purchased from Roche. The sequences of each primer are shown in
High binding surface microplates for ELISA were purchased from Corning Company (Product No. 9018), and a direct ELISA assay was established by human BMP6 antibody (Novus Biologicals Company, DY507) and recombinant BMP6 protein (R&D Systems, 507-BP-020). ELISA was performed according to the manufacturer's protocol.
6
Quantitative PCR Analysis of Gene Expression
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Total RNA was isolated from the treated cells using the Trizol Reagents (Life Technology). The total RNA in each sample was reverse-transcribed into cDNA with PrimeScript RT reagent Kit (Takara BioInc., China). The levels of the transcripts of the target genes were determined using quantitative PCR (qPCR) with SYBR green reagent (Takara BioInc., China) on a Light Cycler PCR system (Roche). GAPDH was used as an internal control for sample normalization. The 2−ΔΔCt method was used to calculate the relative amount of the target mRNA. The primers for mRNA expression are provided in Supplementary Table S5 (Supplementary material 2 ).
7
Quantitative Expression Analysis of CD147 and FUT1
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RMG-I and RMG-I-hFUT cells at an exponential phase of growth were treated with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA; 1 ml per 1×107 cells) to extract total RNA. Complementary DNA (cDNA) was synthesized according to the manufacturer's protocol of the RNA reverse transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: 37°C for 15 min, 85°C for 5 sec, 4°C for 5 min. The primers used were as follows: CD147, forward 5'-GACTGGGTACAAGATCAC-3' and reverse 5'-GCC TCC ATG TTC AGG TTC TCA A-3'; FUT1, forward 5'-AGG TCA TCC CTG AGC TGA AAC GG-3' and reverse 5'-CGC CTG CTT CAC CAC CTT CTT G-3'. The real-time PCR reaction conditions were as follows: Denature at 95°C for 30 sec, 40 cycles of 95°C for 5 sec and 60°C for 30 sec in a 20-µl reaction mixture containing 10 µl SYBR® Premix Ex Taq™ (2X), 0.4 µl PCR forward primer (10 µmol/l), 0.4 µl PCR reverse primer (10 µmol/l), 2 µl cDNA and 7.2 µl dH2O. The Light Cycler PCR system (Roche Diagnostics, Mannheim, Germany) was used for real-time PCR amplification and Cq value detection. The melting curves were analyzed following amplification. All PCR was performed in triplicate. The data were analyzed using the Cq method (20 (link)). The results were considered significant when at least a 2-fold difference in expression levels was detected.
8
Real-Time RT-PCR Analysis of IL-8 and GAPDH
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Total RNAs were converted into cDNAs using Primescript RT master Mix kit (TAKARA). cDNA was incubated with SYBR Green Real-time PCR master mix (Toyobo Co. Ltd., Osaka, Japan) containing 10 pg/ml of the forward and reverse primers, and then amplified using the Light Cycler PCR system (Roche Applied Sciences, Indianapolis, IN, USA). Sequences of the IL-8 primers used were 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ (forward) and 5′-TCTCAGCCCTCTTCAAAAACTTCT-3′ (reverse), generating a 297-base pair PCR product. GAPDH was used as an internal control, the forward primer being 5′-GAAGGTGAAGGTCGGAGT-3′ and the reverse primer being 5′-GAAGATGGTGATGGGATTC-3′, generating a 226-base pair PCR product.
9
Quantifying mRNA Levels in Transduced Cells
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For analysis of relative mRNA expression levels in transduced HUVECs, total RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. After reverse transcription to cDNA using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific), qPCR was performed with the SensiFAST SYBR No-ROX kit (Bioline) and the indicated primers (Supplementary Table S2). The PCR was performed and analyzed either on a LightCycler PCR system (Roche) or a StepOnePlus system (Applied Biosystems). Duplicate reactions were performed for each gene.
For mRNA analysis of mouse lung tissue, lungs were snap-frozen in liquid nitrogen directly after sacrificing the animal. RNA was isolated from 30 mg of snap-frozen lung tissue, using the RNeasy Minikit (Qiagen). Isolated mRNA was used for cDNA synthesis, using iScript reaction mix and iScript reverse transcriptase. cDNA was used for qPCR with the indicated primers (Supplementary Table S2). Reference genes for cultured endothelial cells included GAPDH or TUBB2, while Rps15 was used as a reference gene for qPCR analysis of mouse tissue.
For mRNA analysis of mouse lung tissue, lungs were snap-frozen in liquid nitrogen directly after sacrificing the animal. RNA was isolated from 30 mg of snap-frozen lung tissue, using the RNeasy Minikit (Qiagen). Isolated mRNA was used for cDNA synthesis, using iScript reaction mix and iScript reverse transcriptase. cDNA was used for qPCR with the indicated primers (Supplementary Table S2). Reference genes for cultured endothelial cells included GAPDH or TUBB2, while Rps15 was used as a reference gene for qPCR analysis of mouse tissue.
10
Real-time PCR for EGFR and NEU3 Expression
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Total RNA was prepared using an RNeasy mini kit (Qiagen) and reverse transcribed with SuperScript II (Invitrogen). Real-time PCR was performed with a QuantiTect SYBR Green PCR kit (Qiagen) and a Light Cycler PCR system (Roche). The primers for EGFR and NEU3 were as follows: mouse EGFR sense 5’-TTGGCCTATTCATGCGAA GAC-3’, and antisense 5’-GAGGTTCCACGAGCTCTCTCTCT-3’; human EGFR sense 5’-GACCTCCATGCCTTTGAGAA-3’, and antisense 5’-GCTGACGACT GCAAGAGAAA-3’; mouse NEU3 sense 5’-CTCAGTCAGAGATGAGGATGCT-3’, and antisense 5’- GTGAGACATAGTAGGCATAGGC-3’; and human NEU3 sense 5’-AGGTCAGTCTCCAGTACCTTC-3’ and antisense 5’-ACATCCAGCATCC TGACTGTAG-3’. The expression of glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was determined as an internal control.
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