22Rv1 and C4–2 cells were plated in hormone-deficient media for 72 hours. Following which, cells were treated with vehicle (0nM) or CCS1477 500nM for 5 hours, followed by the addition of 10nM dihydrotestosterone for 3 hours. Chromatin immunoprecipitation (ChIP) was performed as previously described (61 (link)). Briefly, cells were cross-linked with 1% fresh formaldehyde for 10 minutes at room temperature. Chromatin was sheared to 200–700bp using Diagenode Ultrasonicator for 30 cycles. Lysates were incubated with CBP or p300 custom antibody, or AR-FL antibody (Millipore, 06–680). ChIP DNA was extracted via phenol-chloroform method and qPCR was performed using PowerUP SYBR (ThermoFisher). Genes selected for ChIP-qPCR were prioritized based on statistically significant transcriptional downregulation after CCS1477 treatment (1.5 fold change; p=<0.05) and known AR regulation: KLK3, TMPRSS2, FKBP5, ANKRD30B, CHRNA2 (with TFF1 and TGFA representing known CBP binding sites) (48 (link), 49 (link)). Primers were designed at AR target genes using previously described AR binding sites and are listed in Supplementary Table 15 (62 (link)–64 (link)).
Powerup sybr
Manufactured by Thermo Fisher Scientific
Sourced in United States
PowerUp SYBR is a real-time PCR reagent that enables sensitive and reliable detection of DNA targets. It contains a proprietary dye that binds to double-stranded DNA, providing a fluorescent signal for quantification.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Cfx connect qpcr system, by Thermo Fisher Scientific (2 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Micro direct zol purification kit, by Zymo Research (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Quantstudio 5, by Thermo Fisher Scientific (2 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (2 mentions)
Quick rna prep kit, by Zymo Research (2 mentions)
Quick rna microprep kit, by Zymo Research (2 mentions)
Ar fl antibody, by Merck Group (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Rt buffer, by Thermo Fisher Scientific (1 mentions)
Maxima rt, by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3 real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (1 mentions)
15 protocols using powerup sybr
1
ChIP-qPCR Analysis of AR Transcriptional Targets
2
Quantitative PCR Analysis of Gene Expression
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RNA was extracted by homogenizing tissue or cells in TRIzol and then purified using Micro Direct-zol Purification kit following the manufacturer’s instructions (Zymo Research Corporation). RNA was reverse transcribed to cDNA using MultiScribe Reverse Transcriptase (Thermo Fisher Scientific) following the manufacturer’s instructions. qPCRs were run either in duplicates or in triplicates on a CFX Connect qPCR system using PowerUp SYBR (Thermo Fisher Scientific). Gene expression was determined using the ∆∆Ct method (60 (link)) and normalized to mean of Ct values of Hprt1 housekeeping genes. Data are presented as average fold change relative to controls. Primers for specific genes are listed in table S1.
3
Analyzing NIPBL and WAPL Expression in HCT116 Cells
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HCT116 derived RNA was isolated using the RNeasy Plus Kit (Qiagen) according to manufacturer’s instructions. For complementary DNA (cDNA) synthesis, a 50μl reaction containing 20μl RNA, 1500pmol Oligo dT primer (IDT), 1.6mM dNTPs, 1x RT Buffer (Thermo Scientific), 0.5μl RNase OUT (Invitrogen), and 0.7μl Maxima RT (Thermo Scientific) was incubated at 50°C for two hours then at 85°C for 5 min. Samples were stored at -20°C until use. RT-PCR was performed using PowerUP Sybr (ThermoFisher, #A25741) based on manufacturer’s instructions. Briefly, cDNA was diluted to a working concentration of 6μg and HCT116 genomic DNA (gDNA) was diluted in a 1:10 serial dilution. A 6μl reaction was prepared per well, with 1x PowerUP Sybr and 0.2μM of the forward and reverse primers and combined with 4μl diluted DNA. Each reaction was performed in triplicate. qPCR was performed on the QuantStudio7 Flex System. YWHAZ and TBP were used as reference control genes. The sequences of oligonucleotides used for qPCR are: NIPBL forward primer: 5’-TCTCTTTGTTACTTGTCTGTTTCC-3’ and reverse primer 5’-ATGTTTTGCTTTGAAAACCAGTG-3’; WAPL forward primer 5’-GAACTAAAACAGCTCCATCACC-3’ and reverse primer 5’-CACACTTTCAGGCACACCAG-3’; YWHAZ forward primer 5’-CCCGTTTCCGAGCCATAAAAG-3’ and reverse primer 5’-TTTGGCCTTCTGAACCAGCTC-3’; and TBP forward primer 5’-ACAGCTCTTCCACTCACAGAC-3’ and reverse primer 5’-ATGGGGGAGGGATACAGTGG-3’.
4
Amygdalar Gene Expression Quantification
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qPCR was performed as previously described [25 (link), 26 (link)]. RNA was extracted by homogenizing amygdala tissue in Trizol and then purified using Micro Direct-zol Purification kit following manufacturer’s instructions (Zymo). RNA was reverse transcribed to cDNA using MultiScribe Reverse Transcriptase (ThermoFisher Scientific) following manufacturer’s instructions. qPCR reactions were run on a CFX-Connect qPCR system using Powerup SYBR (Thermo Scientific). Changes in expression were determined using the ∆∆Ct method and normalized to mean of Ct values of the Hprt1 housekeeping gene. Data are presented as average fold change relative to controls. Primer sequences have been previously published [26 (link), 33 (link)].
5
Transcriptional Profiling of Autonomic Ganglia in Asthmatic Rats
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Carotid bodies, petrosal ganglia, superior cervical ganglia and nodose ganglia were taken from asthmatic and naïve BN rats (n = 6 ganglia of each type were pooled to increase RNA yield) 3 h after their last aerosol challenge. Total RNA (200 ng confirmed with NanoDrop; Thermo Scientific) was converted to cDNA. PCR amplification was carried out in triplicate (20 μl—1μl mRNA, 7 μl ddH2O, 1 μL primer/each, and 10 μL Powerup SYBR; ThermoScientific) 50 °C/2 min, 95 °C/2 min followed by 45 cycles of 95 °C/3 s, 60 °C/30 s and melt curve (0.1 °C/s; 60-95 °C; QuantStudio 3, Applied Biosystems). Custom primer sequences are listed in Table 1 (Integrated DNA Technologies). Comparisons were made using 2∆ctt with hypoxyribosyltransferase (HPRT) as housekeeping gene [17 (link)].
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Primer sequences (rat tissue)
6
Quantitative Gene Expression Analysis
7
Quantitative RT-PCR for Gene Expression
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Total RNA was isolated from the cells using a FastGene™ RNA Basic Kit (Nippon Genetics, Tokyo, Japan) and reverse‐transcribed into cDNA using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). The SYBR green‐based quantitative polymerase chain reaction was performed using PowerUp SYBR (ThermoFisher Scientific, Waltham, MA, USA) and the QuantStudio 3 Real‐Time PCR System (ThermoFisher Scientific). Relative quantification was performed by the ΔCT method using TBP as the housekeeping gene for normalization. Primer sequences are shown in into Supporting Information.
8
Quantitative Real-Time PCR Protocol
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Total RNA was isolated from cells and tissues by using the RNeasy Mini kit or the RNeasy Plus Mini Kit (Qiagen, Montréal, Québec, Canada), respectively, and stored at −80 °C. Reverse transcription was performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) or the Superscript VILO Kit (Invitrogen), in accordance with the manufacturer's instructions. Gene expression was analyzed by quantitative PCR (qPCR) with SYBR Green chemistry (PowerUp SYBR, ThermoFisher Scientific) on a QuantStudio 6 Real-Time PCR machine (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). All quantitative PCR results were expressed as relative gene expression (rel. exp.), which is the gene of interest normalized to the housekeeping gene, Hprt, by using the 2−ΔC(t) method [19 (link),22 ,33 (link)]. For a complete list of primers and their respective sequences, please see Supplemental Table 2 .
9
Validating Candidate Genes via qRTPCR
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To validate the candidate genes identified in our high-throughput transcriptomic study, total RNA from the inguinal mammary gland was reverse transcribed to cDNA (Biorad iScript cDNA synthesis kit, Hercules, CA), and amplified using quantitative PCR (qRTPCR). Primer sequences for qRTPCR were obtained from Primer bank (Supplementary Table 1 ) [40 (link)–42 (link)], and primers were synthesized by IDT DNA (Coralville, IA). PowerUP SYBR from Applied Biosystems (Foster City, CA) was used to amplify the target genes. Livak’s fold change [43 (link)] was estimated relative to 18S RNA as the house-keeping gene. ANOVA with Tukey post hoc analysis was performed to compare the relative expression between different groups.
10
Mitochondrial Oxidation and Mitophagy Genes Analysis
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Extraction of total RNA was performed using Qiagen tissue extraction Kit (Qiagen, Germantown, MD, USA). Then, cDNA was obtained using high-capacity cDNA Synthesis Kit (Thermo Scientific Co., Waltham, MA, USA). qRT-PCR was carried out using power-up SYBR (Applied Biosystems) utilizing an ABI 7500 RT-PCR System (Applied Biosystems, Foster City, CA, USA). PCR primer pair sequences are shown in Table 1 . Relative expression of the studied mitochondrial oxidation genes, including manganese superoxide dismutase (MnSOD) and uncoupling protein-2 (UCP2), was determined. Moreover, mitophagy-related genes, including phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK 1), Parkin RBR E3 ubiquitin protein ligase (Parkin), FUN14 domain-containing 1 (FUNDC1), unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma 2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) genes, were normalized to the expression level of β-actin gene. The relative quantification was then calculated by the expression 2−ΔΔCt.
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