RNA isolation was performed using TRI Reagent® (T9424, Sigma-Aldrich) according to the manufacturer’s protocol. Retro-transcription PCR was performed using the High Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Ref. 4368814) according to manufacturer’s protocol with a Biometra Tadvanced thermocycler from Analytik Jena AG. For each RNA sample, a negative control lacking the retrotranscriptase in the retrotranscription step was used in qPCR. Gene expression was assessed by qPCR using the LightCycler®96 and the FastStart Essential DNA Green Master mix (Ref. 06402712001) from Roche. Experimental design and gene expression quantification are explained in SM. ATP5F1B, SRP72 and GPI reference genes were chosen after having reviewed literature42 (link),84 (link) and tested their stability in iPS, iRPE, fRPE and iCell RPE. Primer sequences, primer efficiencies, qPCR programmes and additional information on qPCR data processing are available in SM.
Biometra tadvanced thermocycler
Manufactured by Analytik Jena
Sourced in Germany
The Biometra TAdvanced thermocycler is a lab equipment product from Analytik Jena, designed for thermal cycling applications. It is used to accurately control the temperature and cycling parameters for various molecular biology techniques, such as PCR (Polymerase Chain Reaction).
Automatically generated - may contain errors
Lab products found in correlation
Applied biosystems 7900 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (2 mentions)
Nuclease free water, by Thermo Fisher Scientific (2 mentions)
Faststart essential dna green master mix, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Quantitect reverse transcription system, by Qiagen (1 mentions)
Rna isolation kit, by Macherey-Nagel (1 mentions)
Glomax discover microplate reader, by Promega (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
Taqman gene expression assay 20x, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit with rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Multiplex pcr mix, by Qiagen (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay 20, by Thermo Fisher Scientific (1 mentions)
6 protocols using biometra tadvanced thermocycler
1
RNA Isolation and qPCR Analysis
2
Eosinophil RNA Analysis with qPCR
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RNA from eosinophils was purified using an RNA isolation kit (Macherey–Nagel, Düren, Germany). RNA concentrations were normalized, cDNA was synthesized using the QuantiTect Reverse Transcription System (Qiagen, Venlo, Netherlands) in a Biometra TAdvanced Thermocycler (Analytic Jena, Jena, Germany). cDNA was analyzed with GoTaq qPCR Mastermix (Promega, Fitchburg, USA) using the in Supplementary Table 3 listed primers, Morrbid and Bim primers were analogous to Kotzin et al. (2016 (link)). Reactions were performed and analyzed using a LightCycler 480 Instrument II (Roche, Rotkreuz, Switzerland). The 2−ΔΔCt method (Livak and Schmittgen 2001 (link)) was used for the relative quantification of the measured gene expression levels. To ensure comparability among the analysis, the housekeeping gene HPRT was included as internal control and the cell lines HL-60/KG-1 alpha were included as calibrator. Serum IL-5 was measured at baseline using a human IL-5 ELISA Kit (Biorbyt, Cambridge, UK). Reactions were performed and analyzed in a GloMax Discover Microplate Reader (Promega, Fitchburg, USA).
3
RT-qPCR Assay for Slc12a2 and Glpdh
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RNA expression assay was performed for Slc12a2 (Rn00582505_m1) and Glpdh (Rn01775763_g1) genes. High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, USA) was used for reverse transcription reaction in 20 μl reaction volume containing 50 ng of total RNA incubated at 25°C for 10 min, transcripted at 37°C for 120 min, and terminated by heating at 85°C for 5 min using Biometra TAdvanced thermocycler (Analytik Jena AG, Germany). The synthesized cDNA was stored at 4°C until use or at -20°C for longer time. The Real-time PCR was run in triplicate with 4 μl of cDNA template in a 20 μl reaction volume (10 μl of TaqMan Universal Master Mix II, no UNG (Applied Biosystems, USA), 1 μl of TaqMan Gene Expression Assay 20x (Applied Biosystems, USA), and 5 μl of Nuclease-Free Water (Invitrogen, USA) with the program running at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. the reaction was performed using an Applied Biosystems 7900 Fast Real-Time PCR System (Applied Biosystems, USA).
4
Mitominis Protocol for Miseq Sequencing
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The complete Control Region was amplified by ten fragments in two multiplex PCRs using an adjusted version of the mitominis protocol [11 (link)] updated for Miseq Sequencing [12 (link)]. All PCRs were performed on a Biometra Tadvanced thermocycler (Analytik Jena AG, Jena, Germany) using Qiagen Multiplex PCR mix (Qiagen, Hilden, Germany), 0.021–0.24 µM of the various primers [12 (link)] and a 35 cycle PCR of 20 s at 94 °C, 20 s at 55 °C and 20 s at 72 °C. Where possible, 500 copies of mtDNA were used in the PCR.
For library preparation, 1.5 µL of the (unpurified) amplified fragments for each multiplex were used in the KAPA Hyper Prep Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol with the exception that half reaction volumes were used for each step and that the final adapter concentration was 0.2 μM. Libraries were purified using Ampure XP beads (Beckman, Brea, CA, USA) with a 0.6 bead-to-volume ratio and elution in 40 µL water. Libraries were quantified using the KAPA SYBR® FAST qPCR kit as described previously [13 (link)].
Sequencing was performed on the MiSeq FGx (Verogen, San Diego, CA, USA) using v3 chemistry for a minimum read length of 230 bp for each paired-end read; 88–96 samples were pooled in each sequencing run.
For library preparation, 1.5 µL of the (unpurified) amplified fragments for each multiplex were used in the KAPA Hyper Prep Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol with the exception that half reaction volumes were used for each step and that the final adapter concentration was 0.2 μM. Libraries were purified using Ampure XP beads (Beckman, Brea, CA, USA) with a 0.6 bead-to-volume ratio and elution in 40 µL water. Libraries were quantified using the KAPA SYBR® FAST qPCR kit as described previously [13 (link)].
Sequencing was performed on the MiSeq FGx (Verogen, San Diego, CA, USA) using v3 chemistry for a minimum read length of 230 bp for each paired-end read; 88–96 samples were pooled in each sequencing run.
5
RNA Expression Profiling of Slc12a2 and Glpdh
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RNA expression assay was performed for Slc12a2 (Rn00582505_m1) and Glpdh (Rn01775763_g1) genes. High-capacity complementary DNA (cDNA) reverse transcription kit with RNase inhibitor (Applied Biosystems, Carlsbad, CA) was used for reverse transcription reaction in 20 μL reaction volume containing 50 ng of total RNA incubated at 25°C for 10 minutes, transcripted at 37°C for 120 minutes, and terminated by heating at 85°C for 5 minutes using Biometra TAdvanced thermocycler (Analytik Jena AG, Germany). The synthesized cDNA was stored at 4°C until use or at −20°C for a longer time. The real-time polymerase chain reaction (PCR) was run in triplicate with 4 μL of cDNA template in a 20 μL reaction volume (10 μL of TaqMan Universal Master Mix II, no UNG (Applied Biosystems, Carlsbad, CA), 1 μL of TaqMan gene expression assay 20× (Applied Biosystems, Carlsbad, CA), 5 μL of nuclease-free water (Invitrogen, Carlsbad, CA) with the program running at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The reaction was performed using an Applied Biosystems 7900 Fast real-time PCR system (Applied Biosystems, Carlsbad, CA).
6
Multiplex SNP Genotyping Protocol
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Seven pairs of primers for six SNPs were designed using Primer-BLAST and synthesized by Pishgam Biotech or Sinaclon Company (Tehran, Iran). The restriction fragment length polymorphism method was utilized for loci with cleavage sites for restriction endonuclease enzymes. The tetra primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was employed for each GATA and CDX2 loci (Table 1 ).
PCR was conducted on the DNA samples of both patients and controls for all six SNP variants. The PCR was performed in a final volume of 10 μL, including 5 μL of Taq DNA Polymerase 2X Master Mix RED kit (Amplicon, Odense, Denmark), 400 ng DNA template, and 300 nM of each primer in the Biometra TAdvanced thermocycler (Analytik Jena GmbH, Germany). The thermal program included 95 °C for five minutes and then 35 cycles of 95 °C for 25 seconds, 59‒64 °C (depending on the primer) for 20‒25 seconds, and 72 °C for 30 seconds. Then, 72 °C was applied for five minutes as the final extension step. PCR conditions were optimized to prevent nonspecific amplification.
PCR was conducted on the DNA samples of both patients and controls for all six SNP variants. The PCR was performed in a final volume of 10 μL, including 5 μL of Taq DNA Polymerase 2X Master Mix RED kit (Amplicon, Odense, Denmark), 400 ng DNA template, and 300 nM of each primer in the Biometra TAdvanced thermocycler (Analytik Jena GmbH, Germany). The thermal program included 95 °C for five minutes and then 35 cycles of 95 °C for 25 seconds, 59‒64 °C (depending on the primer) for 20‒25 seconds, and 72 °C for 30 seconds. Then, 72 °C was applied for five minutes as the final extension step. PCR conditions were optimized to prevent nonspecific amplification.
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