Genomic DNA was isolated from sorted Lgr5+, crypt, and villus cells using the AllPrep kit (Qiagen) and sonicated to an average size of 150–300 base pairs (bp) (Covaris). DNA fragments were denatured for 10 min at 95°C and immunoprecipitated using 2 µL of anti-5mC (Active Motif) or anti-5hmC (Active Motif) antibody and 10 µL of Protein G beads (Thermo Fisher Scientific) in immunoprecipitation buffer (10 mM sodium phosphate at pH 7.0, 140 mM NaCl, 0.05% Triton X-100). For Me/hMeDIP-qPCR, hydroxymethylated DNA and input DNA were quantified on an Mx3005P qPCR system (Applied Biosystems) using the Brilliant II SYBR Green qPCR master mix (Agilent). qPCR primer sequences are in Supplemental Table S3 . For hMeDIP-seq, DNA fragments were ligated with Illumina adaptors using the NEBNext DNA library preparation master mix set (New England Biolabs). Following adaptor ligation, DNA fragments were denatured and immunoprecipitated using an anti-5hmC antibody (Active Motif). Hydroxymethylated DNA was amplified with adapter-specific primers (12 cycles). Amplified fragments ranging from 150 to 200 bp were size-selected followed by sequencing on a HiSeq2000 (Illumina).
Mx3005p qpcr system
Manufactured by Thermo Fisher Scientific
The Mx3005P qPCR System is a real-time PCR thermal cycler designed for quantitative gene expression analysis. The system features a high-performance optical system and supports a range of detection chemistries, enabling accurate and sensitive quantification of nucleic acid targets.
Automatically generated - may contain errors
Lab products found in correlation
Allprep kit, by Qiagen (2 mentions)
Brilliant 2 sybr green qpcr master mix, by Agilent Technologies (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix reagent, by Thermo Fisher Scientific (2 mentions)
Anti 5hmc, by Thermo Fisher Scientific (1 mentions)
Nebnext dna library preparation master mix set, by New England Biolabs (1 mentions)
Anti 5mc, by Thermo Fisher Scientific (1 mentions)
Protein g beads, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qiaxpert system, by Qiagen (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
6 protocols using mx3005p qpcr system
1
Genome-Wide Hydroxymethylation Profiling
2
Quantitative RT-PCR for Placental Transcripts
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Total RNA (300 ng) from NP and placenta accreta samples PAS was used to analyze the expression of selected mRNAs by reverse transcription using High-Capacity RNA-to-cDNA™ Kit (cat. No. 4368814; Applied Biosystems). Quantitative real-time PCR was performed using TaqMan assays (ERK1, Assay ID: Hs00385075_m1; NFKB1, Assay ID: Hs00765730_m1; AKT1, Assay ID: Hs00178289_m1; PTEN, Assay ID: Hs02621230_s1; STAT3, Assay ID: Hs00374280_m1; TGFB1, Assay ID: Hs00171257_m1; and GAPDH, Assay ID: Hs03929097_g1) and TaqMan Universal PCR Master Mix reagents (cat. No. 4440040; Applied Biosystems). qPCR was run on a Mx3005P qPCR System (Applied Biosystems). mRNA expression was normalized using the 2−ΔCt method relative to GAPDH.
3
Quantitative Real-Time PCR Protocol
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Total RNA and complementary DNA (cDNA) were prepared using the AllPrep kit (Qiagen,) and SuperScript first strand synthesis system (Thermo Fisher Scientific). qRT–PCR reactions were performed on an Mx3005P qPCR system (Applied Biosystems) using Brilliant II SYBR Green qPCR master mix (Agilent). Relative expression levels were determined using comparative Ct values after normalizing to Tbp. qPCR primer sequences are in Supplemental Table S2 . The RNA-seq method was detailed previously (Sheaffer et al. 2014 (link)).
4
Quantification of Antioxidant Gene Expression in HUVEC Cells
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After HUVEC were treated with PETN or DMSO for 24 h RNA was isolated by using TRIzol reagent (Invitrogen) following manufacturer’s instructions. Total RNA concentration was determined using the QIAxpert System (QIAgen). Samples with A260/A280 ratio >1.8 were stored at −80°C until processed. The expression of mRNA levels was determined by reverse transcription using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems). Quantitative real-time PCR was performed using TaqMan assays (HO-1, Assay ID: Hs01110250_m1, eNOS, Assay ID: Hs01574665_m1, superoxide dismutase 2 (SOD2) Assay ID: Hs00167309_m1 and GAPDH, Assay ID: Hs02758991_g1) and TaqMan Universal PCR Master Mix reagents (Applied Biosystems). qRT-PCR was run on a Mx3005P qPCR System (Applied Biosystems). Expressions of all mRNA levels were normalized using the 2−ΔΔCt method relative to GAPDH.
5
RNA Extraction and cDNA Synthesis for qPCR
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Total RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) following the protocol provided by the manufacturer. The cDNAs were synthesized using 2 μg of the extracted RNAs as templates. The reverse transcription reactions were performed using random hex-amer primers and M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA). Then, the cDNAs were detected using the quantitative PCR method in a Mx3005P qPCR System (Applied Biosystems) as described previously [21 (link)]. The CT value greater than 35 was determined to be negative.
6
Regulation of STAT3 and SMAD7 by miR-21-3p
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HTR-8/SVneo and JEG-3 cells were seeded in 6-well plates and allowed to attach overnight to reach 50% confluence at the time of transfection. Transfection was performed for 24 h using Oligofectamine (ThermoFisher) according to the manufacturer's protocol as described before [25] . Sequences mimicking miR-21-3p (ID: MC12979) or a non-genomic scrambled sequence SCR (ID: MH12979) were transfected at a final concentration of 20 nM.
Expression level of STAT3 and SMAD7 was determined 24h after transfection by qPCR. RNA was isolated as described above. 100 ng were reverse transcribed using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, Darmstadt, Germany).
Quantitative real-time PCR was performed using TaqMan assays (STAT3, Assay ID: Hs00374280_m1; SMAD7, Assay ID: Hs00998193_m1 and GAPDH, Assay ID: Hs02758991_g1) and TaqMan Universal PCR Master Mix reagents (Applied Biosystems). qPCR was run on a Mx3005P qPCR System (Applied Biosystems).
Expression of STAT3 and SMAD7 was normalized using the 2 -ΔCt or 2 -ΔΔCt method relative to GAPDH and the respective experimental control.
Expression level of STAT3 and SMAD7 was determined 24h after transfection by qPCR. RNA was isolated as described above. 100 ng were reverse transcribed using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, Darmstadt, Germany).
Quantitative real-time PCR was performed using TaqMan assays (STAT3, Assay ID: Hs00374280_m1; SMAD7, Assay ID: Hs00998193_m1 and GAPDH, Assay ID: Hs02758991_g1) and TaqMan Universal PCR Master Mix reagents (Applied Biosystems). qPCR was run on a Mx3005P qPCR System (Applied Biosystems).
Expression of STAT3 and SMAD7 was normalized using the 2 -ΔCt or 2 -ΔΔCt method relative to GAPDH and the respective experimental control.
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