DNA was extracted from buccal cells and purification of genomic DNA was performed with a standard commercial extraction kit (MagNA Pure LC DNA Isolation Kit I; Roche Diagnostics, Mannheim, Germany) in a MagNA Pure LC System (Roche, Mannheim, Germany). Genotyping of the rs3796863 polymorphism was conducted with a commercial TaqMan® SNP Genotyping Assay (C___1216944_10, Applied Biosystems, Carlsbad, USA) on a Mastercycler® ep realplex (Eppendorf, Hamburg, Germany) according to manufacturers’ standard protocol. Genotyping of SNP rs53576 was conducted using the commercial TaqMan® SNP Genotyping Assay (C___3290335_10, Applied Biosystems, Carlsbad, USA) on a Mastercycler® ep realplex (Eppendorf, Hamburg, Germany) according to manufacturers’ standard protocol.
Mastercycler ep realplex
Manufactured by Eppendorf
Sourced in Germany, United States, China, Japan, Italy, United Kingdom, Canada
The Mastercycler ep realplex is a real-time PCR system designed for efficient and accurate nucleic acid amplification and quantification. It provides precise temperature control and data acquisition for real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (74 mentions)
Rneasy mini kit, by Qiagen (37 mentions)
Trizol, by Thermo Fisher Scientific (30 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (24 mentions)
Sybr premix ex taq, by Takara Bio (21 mentions)
Quantifast sybr green pcr kit, by Qiagen (15 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (15 mentions)
Primescript rt reagent kit, by Takara Bio (13 mentions)
Quantitect reverse transcription kit, by Qiagen (12 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (12 mentions)
Iscript cdna synthesis kit, by Bio-Rad (10 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Primescript rt master mix, by Takara Bio (9 mentions)
Sybr green realtime pcr master mix, by Toyobo (9 mentions)
Sybr premix ex taq kit, by Takara Bio (8 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (8 mentions)
Sybr premix ex taq 2, by Takara Bio (7 mentions)
Rneasy kit, by Qiagen (7 mentions)
Dnase, by Thermo Fisher Scientific (7 mentions)
408 protocols using mastercycler ep realplex
1
Genotyping of SNPs rs3796863 and rs53576
2
Quantitative RT-PCR of HMGA2 and ACTB
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For the relative HMGA2 / ACTB quantification 25 ng total RNA were mixed with SYBR Green, HMGA2 or ACTB specific primers, nuclease free water (Qiagen, Hilden, Germany) and reverse transcriptase according to the “QuantiTect SYBR Green RT-PCR” protocol (Qiagen, Hilden, Germany). The fluorescence of each sample was analyzed in triplicates. As negative controls a non-template and a no-reverse transcriptase control were included. The experiments were performed using the Mastercycler ep realplex (Eppendorf AG, Hamburg, Germany). qRT-PCR conditions were as follows: 30 min at 50°C and 15 s at 95°C, followed by 40 cycles with 15 s at 94°C, 30s at 60°C and 30 s at 72°C. Finally a melting curve analysis was performed to verify specificity and identity of the qRT-PCR products according to the Eppendorf Mastercycler ep realplex instrument instructions. For the comparison of the relative gene expression levels based on the ∆∆CT method the gene expression level of the untreated CT1258 cells was used as calibrator (calibrator expression level was set as 1). Statistical analysis of the qRT-PCR results was done by using the software tool REST 2009, version 2.0.13. A p-value of ≤ 0.05 was considered as statistically significant.
3
Real-time PCR for MDPV Quantification
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Real-time PCR amplification and detection were carried out on Mastercycler ep realplex (Eppendorf, Germany). The concentration of the primers, probe, and templates were optimized based on the obtained fluorescence and lowest threshold cycle (Ct). The optimized TaqMan-based PCR was prepared in a final volume of 25 μl containing 1 μl DNA template, 12.5 μl Premix Ex Taq (Probe qPCR, TaKaRa, Dalian, China), 0.5 μl of each primer (MDPV-qF and MDPV-qR, 10 μmol/l each), 2 μl probe (MDPV-qP, 5 μmol/l), and 8.5 μl Nuclease-free water to to adjust the reaction volume (total reaction volume of 25 μl). The mixed reactions was conducted in a single tube in a Mastercycler ep realplex by using the following thermoprofile: 40 cycles of 95 °C for 5 s, 58 °C for 10 s, and 72 °C for 15 s. 10-fold serial dilutions of plasmid pMD18-NS, containing different copy numbers of DNA (2.97 × 106 to 2.97 × 101 copies/μl) were conducted to generate the standard curve. All of the reactions were conducted in triplicate simultaneously. Analysis of each assay was conducted with CalQplex software (Mastercycler ep realplex, Eppendorf, Germany) according to the instruction manual. The software automatically uses the Ct values of serial dilutions of standards to calculate a standard curve, which shows the Ct values as a function of the amount of different copy numbers of DNA.
4
Quantifying Aβ1-42 mRNA in C. elegans
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Total RNA was isolated from Caenorhabiditis elegans tissues with TRIzol reagent (TIANGEN, Beijing, China). Next, cDNA was synthesized with reverse transcription kit (TIANGEN, Beijing, China) following the manufacturer’s instructions, quantitative real time polymerase chain reaction (qRT-PCR) was performed on the quantitative thermal cycler (Mastercycler ep realplex, Eppendorf, Germany) using SYBR Green agent (TIANGEN, Beijing, China). Primers used to examine the mRNA expression level of Aβ1–42 were 5′-GACGCGGATGCAGAATTCCGA-3′ and 5′-CGCTATGACAACACCGCCCAC-3′. Primers used for qRT-PCR assay to verify the expression profile by RNA-sequencing of C. elegans are listed in the Supplementary Table S1.
5
RT-qPCR Analysis of Gene Expression
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Total RNA was extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA, United States) in accordance with the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of RNA primed by oligo(dT)18 using M-MLV reverse transcriptase (Takara, Kyoto, Japan). The RT-qPCR assay for multiple genes was performed using SYBR® Premix Ex TaqTM II (Takara, Kyoto, Japan). The primer sequences are listed in Supplementary Table S1 . Melting curve analyses were performed for all of the primers. 18S rRNA expression levels were used to normalize the cycle threshold (Ct) (Chandra et al., 2014 (link)). The RT-qPCR was carried out using a Mastercycler ep realplex (Eppendorf, Hamburg, Germany). All the RT-qPCR assays were repeated three times. To assess the extent of RNAi, RNA was extracted from pools of three dsRNA-treated surviving larvae using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), and each treatment was repeated three times. RT-qPCR reactions and data were analyzed in accordance with the methods of Livak and Schmittgen (2001) (link). The data were analyzed using one-way analysis of variance to detect treatment effects compared with the untreated control.
6
Quantifying Vascular Gene Expression
7
Quantitative Real-Time PCR Analysis
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Total RNA was extracted with Trizol reagent (Life Technologies) and 2 μg was reverse-transcribed using Superscript™II (Life Technologies) [22 (link)]. Real-time PCR was performed on a Mastercycler-ep-Realplex (Eppendorf) with primers specific to human and mouse genes (Supplementary Table S2 ) using Quantifast SYBR Green (Life Technologies). PCR was carried out according to the manufacturer's instructions. Melting curve analysis was used to verify that a single peak was obtained for each product with a 95–100% PCR efficiency (Roche software). Relative gene expression levels were normalized according to the Ct value of the housekeeping gene encoding the ribosomal protein L32 and results were expressed as fold differences equal to 2−ΔΔCt.
8
Quantitative RNA Expression Analysis in Bombyx mori
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TRIzol Reagent purchased from Invitrogen was used to extract the total RNA. Subsequently, the RNA was treated with DNase I (Takara) to remove the genome DNA. One microgram of extracted RNA was used as the templates to synthesize cDNA using the RevertAid First-Strand cDNA synthesis kit (Fermentas). Quantitative mRNA measurements were performed with TOYOBO SYBR Green Realtime PCR Master Mix. Three independent biological replicates were examined by Eppendorf Mastercycler ep realplex. The PCR program was the following: incubation at 94°C for 3 min, followed by 40 cycles of 94°C for 10 s and 55°C for 20 s. The standard curves were performed with 10-fold serial dilution cDNA. The data were normalized to B. mori ribosomal protein 49 (Bmrp49) and analyzed by GraphPad Prism version 5.01. All of the qRT-PCR primers are included in Table S1.
9
Gene Expression Analysis by qRT-PCR
10
Quantitative RNA Analysis in Mice
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Total RNA was extracted from mouse tissues and cells using TRIzol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using PrimeScript RT Master Mix
(Takara Bio) following manufacturer’s instructions. cDNAs were amplified using specific set of primers as illustrated inSupplementary
Table 1 for semi-quantitative or real-time RT-PCR. Semi-qRT-PCR products were separated by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining. RT-PCR
was performed by Mastercycler ep realplex (Eppendorf, Hamburg, Germany) using SYBR Premix Ex Taq Kit (Takara Bio). Product of interest was resolved from nonspecific amplification by melt
curve analysis. Gene expression levels were normalized to β-Actin (Actb) and analyzed by 2–ΔΔCt or 2–ΔCt method.
(Takara Bio) following manufacturer’s instructions. cDNAs were amplified using specific set of primers as illustrated in
Table 1
was performed by Mastercycler ep realplex (Eppendorf, Hamburg, Germany) using SYBR Premix Ex Taq Kit (Takara Bio). Product of interest was resolved from nonspecific amplification by melt
curve analysis. Gene expression levels were normalized to β-Actin (Actb) and analyzed by 2–ΔΔCt or 2–ΔCt method.
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