Total RNA from differentiated THP1 cells was isolated using Qiagen spin-columns, contaminating DNA was eliminated by digesting with DNase I (Promega) and RNA was reverse-transcribed using oligo-dT primers and a cDNA synthesis kit according to the manufacturer’s protocol (Promega) as we have previously described [21 (link)–23 (link)]. Duplicate 25 μl real-time PCR reactions were performed in 96 well plates using a SYBR-Green reaction mix (BioRad) and a BioRad iCycler IQ Optical Module according to the supplier’s protocol with the following forward and reverse primers: glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 5’-agcctcgtcccgtagacaaaat and 5’-tggcaacaatctccactttgc; HSP72 (HSPA1A, NM_005345), 5’-ggccagggctggattact and 5’-gcaaccaccatgcaagatta; HSP90 (HSPAA1, NM_005348), 5′-tgcggtcacttagccaagatg and 5′-gaaaggcgaacgtctcaacct; and HSP110 (HSPH1, NM_006644), 5′-gctacacgaattccagctgtga and 5′gagcagcatggtttcgactaaa. Data were quantified using the Gene Expression Ct Difference method and standardized to levels of the housekeeping gene, GAPDH, using Ct values automatically determined by the thermocycler [21 (link)–23 (link)].
Icycler iq optical module
Manufactured by Bio-Rad
Sourced in United States
The iCycler iQ Optical Module is a laboratory instrument designed to measure fluorescence during real-time PCR experiments. It is compatible with the iCycler iQ Real-Time PCR Detection System and provides the necessary optics to perform quantitative fluorescence measurements.
Automatically generated - may contain errors
Lab products found in correlation
Spin column, by Qiagen (1 mentions)
Cdna synthesis kit, by Promega (1 mentions)
Dnase 1, by Promega (1 mentions)
Sybr green reaction mix, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Realmastermix sybr green, by Tiangen Biotech (1 mentions)
Raybio human antibody array c series 1000, by RayBiotech (1 mentions)
2 protocols using icycler iq optical module
1
Quantitative RT-PCR for Heat Shock Proteins
2
Quantitative Analysis of MSC Gene Expression
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Total RNA was isolated from confluent MSCs using the RNeasy mini kit (74104; Qiagen GmbH, Hilden, Germany). cDNA synthesis was performed using ReverTra Ace qPCR RT kit (FSQ-101; Toyobo Co., Ltd., Osaka, Japan) under the conditions: 65˚C for 5 min, followed by 37˚C for 15 min and 98˚C for 5 min. The levels of mRNA of genes of interest were measured by RT-qPCR (iCycleriQ ™ Optical Module, Bio-Rad Laboratories, Inc., Hercules, CA, USA) using RealMaster Mix (SYBR-Green; FP202, Tiangen Biotech Co., Ltd., Beijing, China), under the following conditions: One cycle at 95˚C for 30 sec, 40 cycles at 95˚C for 30 sec, 58˚C for 30 sec and 72˚C for 30 sec, followed by a melt curve from 55 to 95˚C in 0.5˚C increments and 10 sec intervals.
Total amount of mRNA was normalized to endogenous GAPDH mRNA (18) . Sequences of PCR primer pairs are presented in Table I. All experiments were performed three times.
Antibody chip array. Analyzed secretion of protein of supernatant from two groups TLR8-treated and untreated UCMSCs were collected 4 h post stimulation using the RayBio Human Antibody Array C Series 1000 (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer's protocols. Blots were analyzed with ImageJ software version 2.0 (National Institutes of Health, Bethesda, MD, USA).
Total amount of mRNA was normalized to endogenous GAPDH mRNA (18) . Sequences of PCR primer pairs are presented in Table I. All experiments were performed three times.
Antibody chip array. Analyzed secretion of protein of supernatant from two groups TLR8-treated and untreated UCMSCs were collected 4 h post stimulation using the RayBio Human Antibody Array C Series 1000 (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer's protocols. Blots were analyzed with ImageJ software version 2.0 (National Institutes of Health, Bethesda, MD, USA).
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