MDSCs were lysed with PureZol™ (Bio-Rad, Hercules), and RNA was isolated according to the commercial product protocol. iScript™ cDNA synthesis reagents (Bio-Rad, Hercules) were used to generate first strand cDNA according to manufacturer protocol. Real time cycling of reactions that included cDNA diluted ten-fold from above, gene-specific primer probe sets (Applied Biosystems, Foster City), and iQ™ Supermix (Bio-Rad, Hercules) was performed in triplicate using a Step One Plus (Applied Biosystems, Foster City) real time detection system. Gene-specific amplification was normalized to β-actin as an internal reference gene.
Iscript cdna synthesis reagent
Manufactured by Bio-Rad
Sourced in United States
The IScript cDNA synthesis reagents from Bio-Rad are a set of products designed for the reverse transcription of RNA into complementary DNA (cDNA). The core function of these reagents is to facilitate the conversion of RNA into a more stable and easily amplifiable DNA format, which is a crucial step in various molecular biology and genetic analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Gene specific primer probe sets, by Thermo Fisher Scientific (4 mentions)
Iq supermix, by Bio-Rad (4 mentions)
Steponeplus, by Thermo Fisher Scientific (3 mentions)
Purezol, by Bio-Rad (2 mentions)
Nucleospin rna mini kit, by Macherey-Nagel (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Pdgfrβ, by Cell Signaling Technology (1 mentions)
Pdgfrα, by Cell Signaling Technology (1 mentions)
Cyr61, by Cell Signaling Technology (1 mentions)
Phospho c jun ser63, by Cell Signaling Technology (1 mentions)
Recombinant human pdgf bb, by R&D Systems (1 mentions)
Ddit3, by Cell Signaling Technology (1 mentions)
Gdf15, by Cell Signaling Technology (1 mentions)
Runx1, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Antibody to β actin, by Merck Group (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
13 protocols using iscript cdna synthesis reagent
1
Quantitative Gene Expression Analysis of MDSCs
2
Profiling PDGF-BB Signaling Pathways
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Recombinant human PDGF-BB was from R&D Systems (Minneapolis, MN). Antibodies to PDGFRα, PDGFRβ, phospho-PDGFRα/β Tyr849/Tyr857, c-Jun, phospho-c-Jun Ser63, c-Myc, EGR1, RUNX1, DDIT3, CYR61 and GDF15 were from Cell Signaling Technology (Danvers, MA); antibodies to Myb and NFAT5 were from Epitomics (Burlingame, CA); antibodies to SOX5 and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA); antibody to β-actin was from Sigma Aldrich (Sigma Chemical Company, St. Louis, MO); antibody to DIAPH3 was a generous gift from Henry Higgs, Dartmouth Medical School. The c-Myc TF ELISA kit was from Active Motif (Carlsbad, CA). SP600125 and 10048-F4 were from EMD Biosciences (Billerica, MA). iScript cDNA synthesis reagents were from BioRad Laboratories (Hercules, CA). Universal PCR master mix for qRT-PCR and gene-specific assays were from Applied Biosystems (now Life Technologies, Grand Island, NY). Primers for human transcripts were as follows: Hs00171022_m1 for CXCL12; Hs00998500_g1 for CYR61; Hs01107330_m1 for DIAPH3; Hs02758991_g1 for GAPDH; Hs00171132_m1 for GDF15; Hs01110250_m1 for HMOX-1; Hs00998018_m1 for PDGFRA; and Hs01019589_m1 for PDGFRB. Primers for mouse transcripts were Mm00487499_g1 for CYR61; Mm99999915_g1 for GAPDH; Mm00442228_m1 for GDF15; Mm00435546_m1 for Pdgfrb.
3
RNA Extraction, cDNA Synthesis, and qRT-PCR
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RNA extraction, cDNA synthesis and qRT-PCR were carried out as in Kropp et al. (2021a) (link). In brief, RNA was extracted in Trizol Reagent (Thermo Fisher Scientific) and purified with a Zymo QuickRNA MiniPrep kit (Zymo). Synthesis of cDNA was carried out with iScript cDNA Synthesis reagents (Bio-Rad). qRT-PCR was carried out with SYBR Select Master Mix (Thermo Fisher Scientific) on a Bio-Rad CFX96 thermocycler. Primers used in this study are listed in Table S10 .
4
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from cells using the Nucleospin RNA mini kit (#740955, Machery-Nagel) according to manufacturer’s instructions. First-strand cDNA was generated using iScript cDNA synthesis reagents (Bio-Rad, Hercules, CA, USA) using a fixed amount of RNA input (250 ng) in a 10 μL reaction volume. The obtained cDNA was diluted four times and used for transcript analysis.
5
Analyzing Human Macrophage IL-27 Expression
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Human macrophages (2×105/well) cultivated in 24-well dishes were treated as indicated. At appropriate time points, media was removed from cultures, the cells were lysed with PureZol® (Bio-Rad), and RNA was isolated according to commercial product protocol. First strand cDNA synthesis was performed using iScript™ cDNA synthesis reagents (Bio-Rad) according to protocol. For IL-27 p28 and EBI3 gene expression analysis, real time cycling of reactions that included cDNA diluted 20-fold from above, gene-specific primer probe sets (Applied Biosystems), iQ™ Supermix (Bio-Rad) was performed in triplicate using iQ5™ cycler (Bio-Rad). GAPDH was used as an internal reference gene.
6
Quantification of BMDC Gene Expression
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BMDCs were treated with either BCG (MOI 0.5-2.5) or LPS (100 ng/ml) and harvested with TriReagent (Sigma). RNA was isolated by following the manufacturer's protocol for DirectZOL (Zymo Research; Irvine, CA). iScript™ cDNA synthesis reagents (Bio-Rad, Hercules, CA) were used to generate first strand cDNA according to the manufacturer's protocol. Real time cycling of reactions that included cDNA from the above preparation diluted 1:2 in nuclease-free water, gene-specific primer probe sets (Applied Biosystems, Foster City, CA, USA), and iQ™ Supermix (Bio-Rad) was performed in triplicate using a Step One Plus (Applied Biosystems) real time detection system. Gene-specific amplification was normalized to that of actB as an internal reference gene and expressed relative to untreated BMDCs using the formula 2−ΔΔCt.
7
Isolated Cell RNA Extraction and Amplification
8
Measuring Gene Expression in Infected Tissues
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Spleens were homogenized in TRI reagent (Molecular Research Center, Cincinnati, OH). Using the commercial product protocol, the upper aqueous layer following phase separation was mixed with an equal volume of 75% ethanol and transferred to E.Z.N.A. RNA isolation columns (Omega Biotek, Norcross, GA). iScript cDNA synthesis reagents (Bio-Rad, Hercules, CA) were used to generate first-strand cDNA according to the manufacturer’s protocol. Real-time cycling of reactions that included cDNA diluted 15-fold from above, gene-specific primer probe sets (Applied Biosystems, Foster City, CA), and iQ Supermix (Bio-Rad) was performed in triplicate using a StepOnePlus (Applied Biosystems) real-time detection system. Gene-specific amplification was normalized to β-actin as an internal reference gene. Log2-transformed changes in gene expression for infected tissues relative to uninfected control tissues were determined using the formula 2−ΔΔCt. Thus, the data shown reflect the gene expression changes during infection.
9
Quantitative Gene Expression Analysis
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Mouse tissues were snap frozen in liquid nitrogen. RNA was isolated using RNeasy kits (Qiagen) according to manufacturer’s protocol and quantified using Nanodrop spectrophotometer (Thermo Scientific), cDNA generated with iScript cDNA synthesis reagents (Bio-Rad) and duplicate qPCR reactions set up with 5ng template cDNA/RNA, PowerUP SYBR Green Master Mix (Thermo Fisher Scientific) and specific primers (
Table 1 ). Reactions were run on CFX96 Real-Time PCR Detection System (Bio-Rad) with amplification of HPRT as internal housekeeping control. Cycling conditions were: UDG activation, 50°C for 2 min; Dual-Lock DNA polymerase, 95°C for 2 min and 40 cycles of denaturation at 95°C for 15 sec and anneal/extend at 60°C for 30 sec. Gene expression levels were calculated from Ct values using comparative Ct methodology and plotted as relative to the HPRT control.
10
RNA Extraction and cDNA Synthesis
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Total RNA was extracted from cells using the Nucleospin RNA mini kit (# 740955, Machery-Nagel) according to manufacturer's instructions. First strand cDNA was generated using iScript cDNA synthesis reagents (Bio-Rad, Hercules, USA) using a fixed amount of RNA input (250ng) in a 10ul reaction volume. The obtained cDNA was diluted four times and used for transcript analysis.
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