Total RNA was extracted from cells or tumor tissue using an RNeasy kit (Qiagen, Valencia, CA) and reverse transcription was performed using an iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). Cellular levels of IL-8, HIF-1alpha, IL-1beta, IL-6, MCP-1, Par-1, and MMP-1, -2, -3, -9, -13, and -14 mRNA were measured by qRT-PCR using iQSyber Green Supermix (BioRad) on a CFX96 Real Time System C1000 Thermal Cycler. All samples were normalized to B2M and relative fold change was calculated as 2-ΔΔCt. Primer sequences are listed in S1 Table . Cellular levels of CXCL10, CXCR3, and IL-8 were measured by qRT-PCR using Taqman Fast Advanced Master Mix (Life Technologies) and Taqman primers (CXCL10—Hs01124251_g1; CXCR3—Hs01847760_s1; IL-8—Hs00174103_m1; GAPDH—Hs02758991_g1 [Life Technologies]) on a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). All samples were normalized to GAPDH and relative fold change was calculated as 2-ΔΔCt.
Iq syber green supermix
Manufactured by Bio-Rad
Sourced in United States, Italy, Germany
IQ SYBER Green Supermix is a ready-to-use, real-time PCR reaction mix that contains SYBR Green I dye for the detection and quantification of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (11 mentions)
Icycler, by Bio-Rad (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Rneasy kit, by Qiagen (3 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
C1000 thermal cycler, by Bio-Rad (3 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Ncode vilo mirna cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Ncode vilo mirna cdna synthesis kit, by Qiagen (2 mentions)
Superscript double stranded cdna kit, by Thermo Fisher Scientific (2 mentions)
Iscript reverse transcription supermix, by Bio-Rad (2 mentions)
Aurum total rna mini kit, by Bio-Rad (2 mentions)
Iscript kit, by Bio-Rad (2 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Isogen lysis buffer, by Nippon Gene (2 mentions)
Accuprime taq dna polymerase system, by Thermo Fisher Scientific (2 mentions)
Rna genelute kit, by Merck Group (2 mentions)
Myiq real time pcr system, by Bio-Rad (2 mentions)
65 protocols using iq syber green supermix
1
qRT-PCR Analysis of Inflammatory Markers
2
Cardiomyocyte Transcriptional Profiling
3
Chondrocyte and Synovial Cell VEGF-A and PEDF Quantification
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Chondrocytes and synovial cells were harvested at the 3rd, 5th, and 8th days of culture, and RNA was extracted using TRIzol (Invitrogen, Penrose, Auckland, NZ) and quantified with a spectrophotometer. cDNA of the extracted mRNA was synthesized with a SuperSCRIPT First-Strand Synthesis RT-PCR kit (Invitrogen, Penrose, Auckland, NZ) by combining RNA, oligo (dT), and deoxynucleotide triphosphates (dNTPs) and incubating for 5 min at 65℃, then adding 10X RT Buffer, 25 Mm MgCl2, 0.1 M dithiothreitol (DTT), RNaseOUT, and SuperScript and incubating for 50 min at 50℃ and for 5 min at 85℃, then treating with RNase H for min at 37℃. Sequences of the forward and reverse primers of VEGF-A and PEDF are given in Table 1 . Five microliters of the synthesized cDNA, 1 µL of 10 pmol forward and reverse primers, 10 µL of iQ SYBER Green Supermix (Bio-Rad, Hercules, CA, USA), and 3 µL of nuclease-free water were mixed and subjected to 55 cycles of PCR (15 sec at 95℃, 15 sec at 56℃, and 15 sec at 72℃). The results were analyzed with Bio-Rad IQ5 software. The concentrations of mRNA of VEGF-A and PEDF were calculated with reference to a standard curve and compared to the amount of β-actin.
4
Quantitative PCR for Gene Expression
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PCR experiments were performed on total RNA isolated from tissues, treated as described above, using the Triazol reagent (Invitrogen). RNase-free DNase (Ambion, Austin, TX, USA) was used for RNA extraction and cDNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the respective manufacturer’s instructions. Single-strand cDNA samples were obtained using the iScriptcDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) from at least 20 ng of purified RNA. To determine mRNA expression, the following primers were used: (1) Cx36: forward, 5′-ATACAGGTGTGAATGAGGGAGGATG-3′, reverse, 5′-TGGAGGGTGTTACAGATGAAAGAGG-3′72 (link) (2) Actb: forward, 5′-GTGGGGCGCCCCGGCACCA-3′, reverse, 5′-CTCCTTAATGTCACGCACGATTT-3′73 (link) (3) Hprt: forward, 5′-TCCTCATGGACTGATTATGGACA-3′, reverse, 5′-TAATCCAGCAGGTCAGCAAAGA-3′74 (link) (4) Rpl13A: forward, 5′-TCCTCATGGACTGATTATGGACA-3′, reverse, 5′-TAATCCAGCAGGTCAGCAAAGA-3′.74 (link) The new synthesized cDNA was amplified using the oligonucleotide primer listed above, the nucleic acid stain iQ SYBER Green Supermix (Bio-Rad) and an iCycler IQ Real-time PCR System (Bio-Rad).
5
Cdx2null Intestine and Stomach Organoid RNA Analysis
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For RT-PCR analysis, RNA was extracted from Cdx2null SI, control SI and control Sto organoids using the RNeasy Mini RNA Extraction Kit (Qiagen) and reverse-transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Promega). cDNA was amplified in a thermal cycler (Veriti 96 well Thermal Cycler, Applied Biosystems, London, UK). Primers used are listed in Supplementary Materials . For full gel panels see Supplementary Figs 9 and 10 .
For real-time quantitative PCR analysis, cDNA was amplified with iQSyberGreenSupermix (Biorad) on a Biorad CFX Connect Real-Time PCR System. Data were analysed using Biorad CFX Manager Software Version 2.1. Gene expression was normalized according to the expression of the housekeeping gene Gapdh. The primers used are listed inSupplementary Materials .
For real-time quantitative PCR analysis, cDNA was amplified with iQSyberGreenSupermix (Biorad) on a Biorad CFX Connect Real-Time PCR System. Data were analysed using Biorad CFX Manager Software Version 2.1. Gene expression was normalized according to the expression of the housekeeping gene Gapdh. The primers used are listed in
6
Validating Microarray Findings via RT-PCR
7
Quantitative RT-PCR for Gene Expression
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Roots of 10-day-old seedlings or suspension derived protoplasts were harvested, and total RNAs were extracted using the RNeasy kit (Qiagen). For quantitative RT-PCR, RNAs were treated with DNaseI (Ambion) and reverse transcribed using the first-strand cDNA synthesis kit (iScript Advanced cDNA Kit, BioRad). Gene-specific primers and iQSYBER Green Supermix (BioRad) were used on a C1000 Thermal Cycler (BioRad). Quantitative RT-PCR was performed using three replicates, and At1g61670, At3g03210 and At2g32760 were chosen as reference genes [63 (link),64 (link)]. Amplification cycles were analyzed using the Bio-Rad CFX Manager Software (Version 1.6), and fold expression change for each gene of interest was calculated using the ΔΔCt comparative method. Primer sequences are listed in Supplemental Table S1 .
8
Quantitative Gene Expression Analysis
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Total RNA was isolated from BM or endometriotic lesions using TRIzol™ reagent followed by purification using RNeasy® spin columns. qRT-PCR was performed with the cDNA using iQ™ SYBER® Green Supermix (Bio-Rad) with specific primers for each gene. Gene expression was normalized to the expression of GAPDH. Relative mRNA expression was calculated by 2−ΔΔCt method. The primer sequences used are given in Table 1 .
9
Quantitative PCR Analysis of PLCβ Isoforms
10
Quantitative Analysis of Fungal ITS
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Quantitative PCR was carried out to quantify the number of ITS fragments by using LR0R (ACCCGCTGAACTTAAGC) and LR5 primers. The qPCR reactions were performed in triplicate with a solution containing 7.5 μL of qPCR cocktail (iQ SYBER Green Supermix, Biorad, MI, Italy), 1 μL of each primer solution (5 pmol/μL) and 0.1 ng of DNA template in a final volume of 15 μL. The amplifications were carried out by Biorad CFX96 real time PCR detection system.
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