TRI® reagent was purchased from Sigma Aldrich; High-Capacity cDNA Reverse Transcription Kit, Power SYBR Green Supermix and TaqMan® Gene Expression Master Mix from Applied Biosystems; antibodies were purchased from Santa Cruz Biotechnology, USA; ECL reagent (SuperSignal® West Pico Chemiluminescent Substrate) was procured from Thermo Scientific, USA; rabbit anti-cMyc antibody was purchased from Cell Signaling Technology; tissue culture media, serum and additional reagents were procured from HIMEDIA; RNase and cloning kits were purchased from Fermentas; HiPerfect transfection and silencing reagents and QIAprep® Spin Miniprep kit were procured from Qiagen. Restriction enzymes were purchased from Thermo Scientific, USA.
Power sybr green supermix
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Power SYBR Green Supermix is a laboratory reagent designed for real-time quantitative PCR (qPCR) applications. It contains SYBR Green I dye, which binds to double-stranded DNA and emits a fluorescent signal, enabling the detection and quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Merck Group (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Ecl reagent supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cmyc antibody, by Cell Signaling Technology (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rotor gene q series software 1, by Qiagen (1 mentions)
Microdismembrator, by B. Braun (1 mentions)
Tissuemiser, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Bullet blender gold, by Next Advance (1 mentions)
Turbo dna free dnase, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
6 protocols using power sybr green supermix
1
Comprehensive Molecular Technique Protocol
2
Quantitative RT-PCR Analysis of Muscle Transcripts
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Total RNA was isolated from muscle biopsies using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany; including Dnase I treatment) and subsequently the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions. A Micro-Dismembrator (B. Braun Melsungen AG, Melsungen, Germany) was used for tissue disruption and homogenization. A cDNA synthesis with integrated removal of genomic DNA contamination was performed with the QuantiTect Reverse Transcription Kit (Qiagen).
Analysis by qPCR was performed on a Rotor-Gene 2000 real-time PCR thermocycler (Qiagen, Hilden, Germany; program: 40 cycles of 95°C for 10 s followed by 60°C for 45 s) using Power SYBR Green Supermix (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions. Data were quantified using Rotor-Gene Q Series Software 1.7 (Qiagen). Efficiencies were calculated from the slope of template dilution curves with primers for genes of interest (MHC isoforms or genes of energy metabolism) and the reference gene (BSM or RPS12, respectively), and used for quantification of changes of transcript levels by the ΔΔCt-method (Livak and Schmittgen, 2001 (link)). The efficiency of all primer sets was between 95 and 105%.
Analysis by qPCR was performed on a Rotor-Gene 2000 real-time PCR thermocycler (Qiagen, Hilden, Germany; program: 40 cycles of 95°C for 10 s followed by 60°C for 45 s) using Power SYBR Green Supermix (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions. Data were quantified using Rotor-Gene Q Series Software 1.7 (Qiagen). Efficiencies were calculated from the slope of template dilution curves with primers for genes of interest (MHC isoforms or genes of energy metabolism) and the reference gene (BSM or RPS12, respectively), and used for quantification of changes of transcript levels by the ΔΔCt-method (Livak and Schmittgen, 2001 (link)). The efficiency of all primer sets was between 95 and 105%.
3
Quantifying Gene Expression in Mouse Conceptuses
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Total RNA was isolated from GD/ED 10 mouse conceptuses using the RNeasy Plus Mini Kit (Qiagen; Germantown, MD) following homogenization in lysis buffer using the TissueMiser (Fisher Scientific; Waltham, MA) or Bullet Blender Gold (Next Advance; Troy, NY). A minimum of 3 conceptuses were pooled per dam. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, CA). Relative transcript abundance was measured using Power SYBR® Green Supermix (Applied Biosystems; Foster City, CA) with the C1000 Touch Thermal Cycler (CFX96 Real Time Systems; Bio-Rad, Hercules, CA). Each sample was assayed in duplicate for all target and housekeeping genes. Average threshold cycle (Ct) values were normalized to average Ubc78 (link),79 (link) Ct values. Relative transcript abundance of each gene of interest was determined using the ΔΔCt method80 (link). Briefly, transcript expression in individual mice is presented relative to the mean expression value in Mal− mice. When possible, primer pairs were designed to span large introns such that amplification exclusively represented cDNA template. Primer sequences and amplicon sizes are listed in Supplemental Table 1 .
4
Mitochondrial Gene Expression Analysis
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Total RNA was isolated using the Qiagen RNeasy Mini Kit (QIAGEN) and then treated with TURBO DNA-free DNase (Ambion) to remove contaminating genomic DNA. Reverse transcription of RNA was performed using the M-MLV transcriptase system (Invitrogen) and random hexamer primers.
Gene expression analysis via qRT-PCR was carried out using Power SYBR Green Supermix (Applied Biosystems) running on an ABI Prism 7000 sequence detection system (Applied Biosystems). Primer sets were designed as described previously [31] (link) (Table S2 ) to monitor the expression of genes involved in the splicing of junctions within mitochondria.
Gene expression analysis via qRT-PCR was carried out using Power SYBR Green Supermix (Applied Biosystems) running on an ABI Prism 7000 sequence detection system (Applied Biosystems). Primer sets were designed as described previously [31] (link) (
5
Quantitative Analysis of LMP1 mRNA Expression
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Quantitative RT-PCR was performed to measure LMP1 mRNA levels in transduced AD293 cells. A total of 1x106 AD293 cells were transduced with 1x106 CFU Ad5-LMP1 with or without 1ug/ml doxycycline added to the culture media at the time of transduction. AD293 cells were transfected with pcDNA3.1-LMP1 plasmid as a positive control using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. After 48 hours, total RNA was prepared from cells using the RNeasy kit (Qiagen), and reverse transcribed in a 20 μl reaction containing 0.1 μg of total RNA, 0.1 μg of oligo(dT), 200 U of reverse transcriptase (Finnzymes) and 0.2 μM each of dATP, dCTP, dGTP and dTTP. After 1 hr incubation at 40°C, cDNA products were generated. Real-time PCR then was performed using the Power SYBR Green Supermix (Applied Biosystems) with primers specific to LMP1. For normalization, GAPDH and β-actin real-time PCR was carried out on the same samples. Normalized mRNA levels for each transcript were calculated as (1/2ΔCt × 1,000), where ΔCt value = Ct (test mRNA)—Ct (GAPDH mRNA). To control for contamination with genomic DNA, parallel amplifications were performed in the absence of reverse transcriptase. These were uniformly negative.
6
Mitochondrial RNA Splicing and Stability Analysis
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Total RNA was isolated from 0.01-g 14-day-old wild-type, homozygous ppme−/−, and complemented seedlings with a Qiagen RNeasy Mini Kit (QIAGEN). Then, cDNA was synthesized with M-MLV reverse transcriptase (Invitrogen) and random hexamer primers as previously described.10 (link) The RNA splicing efficiencies of mitochondrial genes were evaluated using 2 strategies. Primers targeting either the exon-exon or exon-intron junctions of individual introns in mitochondrial genes were used for amplification. Then, the amplicons for these junctions were detected using Power SYBR Green Supermix (Applied Biosystems) and an ABI Prism 7000 sequence detection system (Applied Biosystems). The spliced/unspliced ratio was calculated from the ratio of the quantity of exon-exon junctions to the quantity of exon-intron junctions, as previously described.10 (link) The spliced products were further amplified by RT-PCR with the primer sets targeting the exon-exon junctions; PCR was performed for 35 cycles to saturation, and any unspliced products were detected. Mitochondrial RNA stability was evaluated using primer sets as previously described.9 (link) All quantifications were normalized to 4 housekeeping genes: YSL8, RPL5B, UBC, and TUB6.
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