Mastercycler realplex2

Manufactured by Eppendorf

Sourced in Germany, United States

The Mastercycler Realplex2 is a real-time PCR system designed for high-precision gene expression analysis. It features precise temperature control, a large sample capacity, and advanced software for data analysis and result interpretation.

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198 protocols using mastercycler realplex2

Quantitative Analysis of YFP Expression in Plants

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For Quantitative Real-Time PCR (qPCR), young leaves from 12-week-old plants grown in greenhouse at normal ambient temperature were harvested and frozen in liquid nitrogen for further experiments. Total RNA was extracted with the E.N.Z.A. Plant RNA Kit (OMEGA Bio-tek, GA, United States) and treated with DNase I (Thermo Scientific, MA, United States). 1 μg of RNA was used for cDNA synthesis with the RevertAid first-strand cDNA synthesis kit (Thermo Scientific, MA, United States). qPCRs were performed with an Eppendorf realplex² Mastercycler (Eppendorf, Hamburg, Germany) using the SensiFAST SYBR Lo-ROX Mix (Bioline, London, United Kingdom) and the primers YFP forward (5′-AAGCAGAAGAACGGCATCAA-3′) and YFP reverse (5′-GGGGGTGTTCTGCTGGTAGT-3′), to amplify parts of YFP. Data were normalized to SlACT and samples were measured in at least three biological and four technical replicates.

Shuai H., Chen T., Wlk T., Rozhon W., Pimenta Lange M.J., Sieberer T., Lange T, & Poppenberger B. (2022). SlCESTA Is a Brassinosteroid-Regulated bHLH Transcription Factor of Tomato That Promotes Chilling Tolerance and Fruit Growth When Over-Expressed. Frontiers in Plant Science, 13, 930805.

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Expression analysis of MoOeIF3K and complex

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The strains were cultured in liquid CM for 4 days at a speed of 110 rpm. The mycelia were filtered, washed with sterilized ddH₂O, dried with absorbent paper, and further dry frozen in liquid nitrogen. Total RNA was extracted from the individual strains using a HiPure Universal RNA kit (R4130-02; Magen, China). The expression of MoOeIF3K under different stress conditions and expression of individual subunits of the MoeIF3 complex in ΔMoOeif3k were monitored by quantitative real-time PCR (qRT-PCR) assays. Reverse transcription of RNAs was performed using the PrimeScript RT regent Kit with gDNA Eraser (RR047A; Takara, Japan). A 10-μl reaction mix was formulated as follows: 5 μl TB green, 3.4 μl RNase free water, 0.3 μl of each 10 μM forward and reverse primers listed in Supplementary Table 1 and 1 μl cDNA template. qRT-PCR was carried out with Eppendorf Realplex2 master cycler (AG 223341; Eppendorf, Hamburg, Germany). The raw qRT-PCR data were analyzed using the formula delta delta-CT (2^–ΔΔCT) method described by Rao et al. (2013) and Abdul et al. (2018) (link). The expression level of tubulin was used as the reference or internal control. Error bars represent mean ± SD. The data were obtained from three independent biological experiments with three technical replicates for each independent experiment.

Lin L., Cao J., Du A., An Q., Chen X., Yuan S., Batool W., Shabbir A., Zhang D., Wang Z, & Norvienyeku J. (2021). eIF3k Domain-Containing Protein Regulates Conidiogenesis, Appressorium Turgor, Virulence, Stress Tolerance, and Physiological and Pathogenic Development of Magnaporthe oryzae Oryzae. Frontiers in Plant Science, 12, 748120.

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Age and Sex-Dependent Gene Expression

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Three independent biological replicates of young (3-5 days) and old (45-50 days) males and females of each genotype was collected separately. Total RNA was extracted from whole body homogenates (25 flies) using Tri Reagent (Sigma, St. Louis, MO, USA). Samples were treated with Takara Recombinant DNase I (Clontech Laboratories Inc., Mountain View, CA, USA) followed by cDNA synthesis with iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed on the Eppendorf realplex² Mastercycler (Eppendorf, Hauppauge, NY, USA) under default thermal cycling conditions, with a dissociation curve step. Every reaction contained Power SYBR Green (Applied Biosystems, Carlsbad, CA, USA), 10 ng cDNA, and 400 nM primers. Primer sequences are provided in Supplemental Table S1. Data were analyzed using the 2^−ΔΔCT method with mRNA levels normalized to the housekeeping gene rp49 (Hanna et al., 2015 (link)). Relative mRNA expression was calculated with respect to pooled data from young male wild type w¹¹¹⁸ and Canton-S flies set as 1.

Bednářová A., Hanna M.E., Rakshit K., O'Donnell J.M, & Krishnan N. (2017). Disruption of dopamine homeostasis has sexually dimorphic effects on senescence characteristics of Drosophila melanogaster. The European journal of neuroscience, 45(6), 816-825.

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Quantifying Promoter DNA Methylation

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DNA was directly extracted from lung samples according to manufacturer’s instructions using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Percent CpG promoter methylation was then measured using the EpiTect Methyl II PCR Assay (Qiagen, Hilden, Germany). Briefly, 250 ng of isolated DNA was incubated in 26 μL of 5 × restriction digestion buffer and methylation sensitive/dependent/null enzymes overnight at 37°C, and then heat inactivated for 20 minutes at 65°C. Ninety-six-well plates were prepared with 12.5 μl of SYBR Green qPCR Master mix, 6.5 μl of RNase-free water, 1 μl of the corresponding EpiTect PCR Primer, and 5 μl of DNA digest. The DNA was then amplified with 40 cycles of 97°C (denaturation) for 15 s and 72°C (annealing and elongation) for 1 min using the Eppendorf Realplex2 Mastercycler (Eppendorf, Westbury, NY). The fraction of methylated DNA for each gene promoter was calculated by normalizing the DNA amount to the amount of digestible DNA. The amount of digestible DNA was equal to the total amount of DNA (determined from the mock digest) minus the amount of DNA resistant to DNA digestion (determined from the double digest).

Kalash R., Berhane H., Au J., Rhieu B.H., Epperly M.W., Goff J., Dixon T., Wang H., Zhang X., Franicola D., Shinde A, & Greenberger J.S. (2014). Differences in Irradiated Lung Gene Transcription Between Fibrosis-Prone C57BL/6NHsd and Fibrosis-Resistant C3H/HeNHsd Mice. In vivo (Athens, Greece), 28(2), 147-171.

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Quantifying Gene Expression in Stress-Exposed Flies

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Three independent bio-replicates of male flies (6-8 days old) were collected following 4 h exposure to HP stress from each genotype. In parallel, flies from untreated groups were also collected in a similar manner. Total RNA was extracted from whole body homogenates of flies (25) using Tri Reagent (Sigma, St. Louis, MO, USA). The samples were purified and treated with Takara Recombinant DNase I (Clontech Laboratories Inc., Mountain View, CA, USA). Synthesis of cDNA was achieved with the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed on the Eppendorf realplex² Mastercycler (Eppendorf, USA) under default thermal cycling conditions, with a dissociation curve step. Every reaction contained Power SYBR Green (Applied Biosystems), 10 ng cDNA, and 400 nM primers. Primer sequences are given in Supplemental Table T1. Data were analyzed using the 2^−ΔΔCT method with mRNA levels normalized to the gene rp49. Relative mRNA amplitude was calculated with respect to untreated control wild type w¹¹¹⁸, or wild type Canton-S flies whose expression for a particular gene was set as 1.

Hanna M.E., Bednár̂ová A., Rakshit K., Chaudhuri A., O’Donnell J.M, & Krishnan N. (2015). Perturbations in dopamine synthesis lead to discrete physiological effects and impact oxidative stress response in Drosophila. Journal of insect physiology, 73, 11-19.

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Gene Expression Analysis of Macrophages

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TRIzol (Invitrogen) along with the RNeasy^™ Mini Kit (Qiagen) were used to extract and purify total RNA from lysed macrophage cells prior to reverse transcription using a First-strand cDNA Synthesis System (Marligen Bioscience, MD). Fast Start Universal SYBR Green Master (Rox) (Roche Applied Science) was used to perform quantitative real-time PCR on an Eppendorf Realplex2 Mastercycler (Eppendorf, Hamburg, Germany). The following primers were used: Mouse 18S rRNA (internal control), 5’-CGCGGTTCTATTTTGTTGGT-3’ (forward) and 5’-AGTCGGCATCGTTTATGGTC-3’ (reverse); Mouse TNFα, 5’-CGTCAGCCGATTTGCTATCT-3’ (forward) and 5’-CGGACTCCGCAAAGTCTAAG-3’ (reverse); Mouse IL-6, 5’-AGTTGCCTTCTTGGGACTGA-3’ (forward) and 5’-TCCACGATTTCCCAGAGAAC-3’ (reverse); Mouse IL-1α, 5’-ATGGCCAAAGTTCCTGACTTG-3’ (forward) and 5’-TGACGTTTCAGAGGTTCTCAG-3’ (reverse); Mouse IL-1ß, 5’-GCCCATCCTCTGTGACTCAT-3’ (forward) and 5’-AGGCCACAGGTATTTTGTCG-3’ (reverse). The amplification program was 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 68 °C for 20 s. Finally, a melting curve analysis from 60 °C to 95 °C every 0.2 °C was performed. Gene abundances were calculated by relative quantification and normalized to the internal control, 18S rRNA.

Hou L., Tian H.Y., Wang L., Ferris Z.E., Wang J., Cai M., Older E.A., Raja M.R., Xue D., Sun W., Nagarkatti P., Nagarkatti M., Chen H., Fan D., Tang X, & Li J. (2022). Identification and biosynthesis of pro-inflammatory sulfonolipids from an opportunistic pathogen Chryseobacterium gleum. ACS chemical biology, 17(5), 1197-1206.

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Genotyping of rs4042056 Using KASP Assay

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The genotype of the rs4042056 was determined with the KASP method (LGC Genomics GmbH, Berlin, Germany), using a customized assay on a realplex² Mastercycler (Eppendorf AG, Hamburg, Germany), followed by fluorescence end-point reading with an Applied Biosystems 7800 Real-Time PCR system (Life Technologies GmbH, Darmstadt, Germany). For each sample, 10 ng of genomic DNA was used and mixed with KASP reagents according to the manufacturer. The KASP assay contained the allele-specific HEX-conjugated primer 5′-ggc tct tct gcg tga agc-3′, the FAM-conjugated primer 5′-ggc tct tct gcg tga agt-3′, and the common primer 5′-cag gtg gca gca ggg gaa ca-3′.

Recke A., Konitzer S., Lemcke S., Freitag M., Sommer N.M., Abdelhady M., Amoli M.M., Benoit S., El-Chennawy F., Eldarouti M., Eming R., Gläser R., Günther C., Hadaschik E., Homey B., Lieb W., Peitsch W.K., Pföhler C., Robati R.M., Saeedi M., Sárdy M., Sticherling M., Uzun S., Worm M., Zillikens D., Ibrahim S., Vidarsson G, & Schmidt E. (2018). The p.Arg435His Variation of IgG3 With High Affinity to FcRn Is Associated With Susceptibility for Pemphigus Vulgaris—Analysis of Four Different Ethnic Cohorts. Frontiers in Immunology, 9, 1788.

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qPCR Analysis of CTGF Expression

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RNA was extracted using trizol/chloroform isolation followed by Ambion mirVana kit (AM1560). Total RNA was reverse-transcribed using random primers (Amersham Biosciences), and β-actin primers were used to control for cDNA concentration in a separate PCR reactions for each sample. LightCycler Fast Start DNA Master SYBR Green Mix (Roche) was added to each PCR reaction along with cDNA and 1 pmol primer in a total volume of 10 µl. qPCR was performed on a eppendorf realplex2 mastercycler using the real-time primers provided, according to instructions. Ct values were converted to fold expression changes (2^−ΔΔCt values) following normalization to β-actin. Primer sequences are listed below.

Gene	Sequence

CTGF	F 5’-GTGAGTCCTTCCAAAGCAGC-3’
	R 5’-TAGTTGGGTCTGGGCCAAAT-3’

β-actin	F 5’-GTGGGCCGCCCTAGGCACCA-3’
	R 5’-CGGTTGGCCTTAGGGTTCAGGG-3’

Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.

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Profiling Prostate Cell Transcripts

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Total RNA was extracted from control (n = 6) or Nkx3-1^Cre/+;Gata3^flox/flox (n = 6) total or sorted prostate cells using an RNeasy mini kit (Qiagen) and reverse transcribed with Moloney murine leukemia virus (Invitrogen) according to the manufacturer’s protocol. Real-time qPCR was performed using Green-2-go Mastermix (BioBasic) on a Realplex2 Mastercycler (Eppendorf). All primers used are listed in Table S2.

Shafer M.E., Nguyen A.H., Tremblay M., Viala S., Béland M., Bertos N.R., Park M, & Bouchard M. (2017). Lineage Specification from Prostate Progenitor Cells Requires Gata3-Dependent Mitotic Spindle Orientation. Stem Cell Reports, 8(4), 1018-1031.

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Quantitative Real-Time PCR Analysis

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Real-time RT-PCR experiments were performed using the Realplex² Mastercycler (Eppendorf). Primers were designed using Primer Express® software (Applied Biosystems, Supplemental Information). RNA was prepared using Trizol, and cDNA was synthesized using the Superscript system (Invitrogen, Carlsbad, CA). Samples were analyzed in duplicate and normalized to 18S and Gpi1 expression levels. We also custom designed Taqman 384-well expression (Applied Biosystems): Dtl (Mm00712787_m1), Hells (Mm00468580_m1), Gfap (Mm01253033_m1), Mcm2 (Mm00484804_m1), Rrm2 (Mm00485881_g1), Tcf19 (Mm00508531_m1), Top2a (Mm00495703_m1), Uhrf1 (Mm00477873_g1). 500ng of sample RNA was used for cDNA synthesis (High Capacity RNA-to-cDNA kit; Applied Biosystems #4387406).

Benavente C.A., Finkelstein D., Johnson D.A., Marine J.C., Ashery-Padan R, & Dyer M.A. (2014). Chromatin remodelers HELLS and UHRF1 mediate the epigenetic deregulation of genes that drive retinoblastoma tumor progression. Oncotarget, 5(20), 9594-9608.

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