Total RNA (10 μg) was incubated with 2 U DNase I (Roche) for 30 min at 37°C to remove any contaminating DNA. First strand cDNA was synthesised from 1 μg of total RNA using 200 U SuperScript™ III Reverse Transcriptase (Invitrogen) and 250 ng random p(dN)6 primers (Roche) following the manufacturer's instructions. Levels of eRF1 mRNA were measured in an Applied Biosystems 7500 Fast Real-Time PCR System using ABsolute™ QPCR Low ROX Mix (ABgene) and a TaqMan® Gene Expression Assay (ETF1 (eRF1) Gene Assay ID: Hs01107358_g1; GAPDH Gene Assay ID: Hs99999905_m1 Applied Biosystems). 18S rRNA was measured as a reference for relative quantification using TaqMan® Ribosomal Control Reagents (Applied Biosystems).
Absolute qpcr low rox mix
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Absolute qPCR low ROX mix is a ready-to-use solution for quantitative polymerase chain reaction (qPCR) experiments. It contains all the necessary components, including a DNA polymerase, buffer, and dNTPs, to perform accurate and reliable qPCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Absolute qpcr sybr low rox mix, by Thermo Fisher Scientific (2 mentions)
High capacity reverse transcription kit with ribonuclease inhibitor, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Random p dn 6 primers, by Roche (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Realtime qpcr assay tool, by Integrated DNA Technologies (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
Random primer, by Roche (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
5 protocols using absolute qpcr low rox mix
1
Quantification of eRF1 mRNA Expression
2
Quantifying Gene Expression in HT22 Cells
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We extracted total RNA from HT22 cells or mouse brain (hippocampal region; see [18 (link)] for method) using the TRIzol reagent (Invitrogen) following the manufacturer’s instructions. We treated total RNA with DNase 1 (20U; Roche, Basel, Switzerland) to remove contaminating genomic DNA and conducted reverse transcription with 1 μg RNA using the High Capacity Reverse Transcription kit with ribonuclease inhibitor (Applied Biosystems, Life Technologies Corp, Foster City, CA). For RTqPCR we used Taqman assays for Gapdh and Klf9 [7 (link)] and SYBR green assays for all other genes. All oligonucleotide primer sequences are given in Additional file 18 : Table S9. We conducted RTqPCR using an ABI 7500 fast real-time PCR machine with Absolute qPCR low ROX mix (for Taqman assays) or Absolute qPCR SYBR low ROX mix (ABgene, Epsom, UK). We designed SYBR green assays using Integrated DNA Technology’s RealTime qPCR Assay tool; where possible we designed assays to span exon-exon boundaries. We used a relative quantitation method using serial dilutions of a cDNA pool to generate standard curves. We normalized all genes to the reference gene Gapdh whose mRNA was unaffected by treatments (data not shown.)
3
RNA Extraction and RT-qPCR Analysis
4
Quantifying mRNA Expression in Biomedical Samples
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Total RNA was isolated from the serum of patients with breast cancer, mouse heart, and H9C2 cells using TRIzol reagent (Invitrogen, ThermoFisher Scientific, MA, USA). The cDNA was synthesized using the TaqMan™ Reverse Transcription Reagents (Invitrogen). Then, qPCR was carried out using the ABsolute qPCR low ROX Mix (ThermoFisher Scientific) on the ABI PRISM 7500 system according to the manufacturer’s protocol. The reaction mixture contains Absolute blue SYBP Green ROX (12.5 μL), forward primer (1.75 μL), reverse primer (1.75 μL), cDNA (2 μL), and nuclease-free water (7 μL). The reaction conditions were 95°C for 15 min, 40 cycles of 95°C for 15 s, 56°C for 30 s, and 72°C for 30 s. GAPDH served as the control. The mRNA expression was calculated by the 2−ΔΔCt method. The primer sequences are shown in Table 2 .
5
RNA Extraction and qRT-PCR Analysis
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Total RNA from transfected cells was isolated using TRIzol® (Invitrogen) according to the manufacturer’s instructions. Total RNA (10 μg) was incubated with 2 U DNase I (Roche) for 30 min at 37°C (25 m M Tris-HCl pH 7.2, 5 mM MgCl2, 0.1 m M EDTA), and DNase-treated and untreated RNA samples were analysed on formaldehyde agarose gels to determine quality and integrity. cDNA synthesis used total RNA (1 μg) with 250 ng random primers (Roche) and 200 U SuperScript™ III reverse transcriptase (Invitrogen). eRF1 and 18S rRNA transcript levels were determined using 2.5 ng of cDNA with a TaqMan® Gene Expression Assay (eRF1 assay Hs01107358_g1, Applied Biosystems) or TaqMan Ribosomal Control Reagents (18S rRNA, Applied Biosystems). Reactions were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems). TaqMan reactions were performed with ABsolute QPCR Low ROX Mix (Thermo Fisher Scientific). Expression levels were calculated using the comparative CT method of relative quantification, after confirming that amplification efficiency of the target gene was within 5% of the reference gene efficiency.
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