Total RNA was isolated using the Absolute RNA Kit from Stratagene (Agilent Technologies) and cDNAs were synthesized using SuperSript III First strand cDNA synthesis system for RT-PCR (Invitrogen) according to manufacturer’s instructions. qPCR was performed using the ABI7500 Prism SDS Real-Time PCR Detection System (Applied Biosystems) with a SYBR Green PCR Master Mix kit (Applied Biosystems) and a standard temperature protocol. The results are expressed as relative quantities and calculated using the 2-ΔΔCT method. Three separate qPCR experiments were performed. Actin was used as a control gene for normalization. Primers were purchased from Qiagen (QuantiTect primer assay, IL-33, IL-8, IL-6, SELE) or Sigma Genosys (Actin, NFKB2, ICAM1, VCAM1, ST2).
Abi7500 prism sds real time pcr detection system
Manufactured by Thermo Fisher Scientific
The ABI7500 Prism SDS Real-Time PCR Detection System is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It is capable of performing quantitative and qualitative real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Absolute rna kit, by Agilent Technologies (1 mentions)
Sybr green pcr master mix kit, by Thermo Fisher Scientific (1 mentions)
Random hexamer oligonucleotides, by Thermo Fisher Scientific (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Ozyme (1 mentions)
Primer express software, by Merck Group (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using abi7500 prism sds real time pcr detection system
1
Quantitative Gene Expression Analysis
2
Quantitative RNA Expression Analysis
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Total RNA was extracted using ready-to-use TRIzol Reagent (Ambion, Life Technologies) and purified with RNeasy Mini kit (Qiagen) following manufacturer's instructions. Complementary DNA was reverse transcribed from 1 μg total RNA with Moloney murine leukemia virus reverse transcriptase (Sigma) using dNTP (Promega) and random hexamer oligonucleotides (ThermoFisher) for priming. qPCR was performed using SYBR green Supermix (OZYME) in an ABI7500 Prism SDS Real-Time PCR Detection System (Applied Biosystems). The mRNA content was normalized to β-actin mRNA and quantified using the 2-∆∆Ct method. Primers used for cDNA amplification were purchased from Sigma and are listed in Supplemental Table 1.
3
Transcriptomic Analysis of Tumor-Associated Endothelial Cells
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TA-HECs and TA-ECs from MCA reg or MCA prog tumors pooled from 3 mice, and LN-HECs and LN-ECs from peripheral LNs pooled from 3 mice, were sorted on a BD influx into low binding tubes containing 350 mL of RLT Buffer (QIAGEN, 74104) supplemented with 1% b-mercaptoethanol. Total RNA was isolated using an RNeasy Mini Kit (QIAGEN, 74104) and cDNA were synthesized using SuperScript" VILO cDNA Synthesis Kit according to the manufacturer's instructions (ThermoFisher Scientific, 11754-050) . qPCR was performed using the ABI7500 Prism SDS Real-Time PCR Detection System (Applied Biosystems) with Power SYBR Green PCR Master Mix (ThermoFisher Scientific, 4367659) and a standard temperature protocol. The results are expressed as relative quantities and calculated using the 2-DDCT method. Genes were analyzed in triplicate, and two to four separate qPCR experiments were performed for each gene. Primers were designed using Primer Express software and were purchased from Sigma-Aldrich. Hprt was used as a control gene for normalization.
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