To obtain the CHS and UFGT sequences, a polymerase chain reaction (PCR) analysis was performed in a MultiGene thermal cycler (Labnet International, Inc., Woodbridge, NJ, US) using 20 ng template DNA, 0.5–1 U Taq (Bernardo Scientific Corp., Taipei, Taiwan), 100 μM deoxyribonucleotide triphosphate, and 0.2 μM of each primer. Primers for CHS amplification were adapted from those used by Chiang et al. [19 (link)]. The primers for UFGT amplification were designed from the transcriptomic assembly of inflorescences buds used by Huang et al. [15 (link)]. The primer sequences for UFGT were ScUGT-F1: 5′-GTTCCAATGATAGCTCATGG-3′ and ScUGT-R1: 5′-GGAACATAGGCACTCAATTC-3′. The PCR program was set to 94 °C for 3 min for enzyme activation, followed by 35 cycles at 94 °C for 40 s, melting temperature for 40 s, and 72 °C for 80 s, with a 5-min final extension at 72 °C. The PCR products were sequenced directly in both directions using an ABI BigDye 3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). All sequences were visually checked via chromatograms using an ABI PRISM®3730XL DNA Sequencer (Perkin-Elmer, Foster City, CA, USA).
Multigene thermal cycler
Manufactured by Labnet
Sourced in United States
The MultiGene thermal cycler is a laboratory instrument used for the amplification of DNA samples through the Polymerase Chain Reaction (PCR) process. It precisely controls the temperature and duration of the various steps involved in the PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Abi bigdye 3.1 terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 3730xl dna sequencer, by PerkinElmer (1 mentions)
High capacity cdna synthesis kit, by Roche (1 mentions)
Sensifast sybr green mix, by Meridian Bioscience (1 mentions)
Dnase 1 treatment, by Roche (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Qscripttm cdna synthesis kit, by Quanta Biosciences (1 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions)
Real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
No ung, by Thermo Fisher Scientific (1 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
7 protocols using multigene thermal cycler
1
PCR Amplification of CHS and UFGT Sequences
2
Quantitative RT-PCR Analysis of Liver Transcripts
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RNA was extracted from frozen liver tissue using ice-cold TRIzol reagent (Life Technologies) according to the manufacturer's protocol, followed by DNase I treatment (Roche). Complementary DNA was synthesized by standard reverse transcription (High Capacity cDNA synthesis kit, Roche) on a MultiGene thermal cycler (Labnet). Amplification and semi-quantification of transcripts were performed using Sensifast SYBR Green mix (Bioline) on a real-time PCR system (iCycler, Bio-Rad) with mouse gene-specific primers (Integrated DNA Technologies). Cyclophilin A (CypA) was used as a housekeeping gene. Primers used were CypA (F: 5′-CGATGACGAGCCCTTGG-3′, R: 5′-TCTGCTGTCTTTGGAACTTTGTC-3′), alpha-fetoprotein (Afp; F: 5′-CCCGCTTCCCTCATCC-3′, R: 5′-GAAGCTATCCCAAACTCATTTTCG-3′), glucose-6-phosphate dehydrogenase (G6pd; F: 5′-AAGAAGCCTGGCATGTTCTT-3′, R: 5′-GAAGCCCACTCTCTTCATCA-3′), lipoprotein lipase (Lpl; F: 5′-GGATGGACGGTAACGGGAAT-3′, R: 5′-ATAATGTTGCTGGGCCCGAT-3′), Cd36 (F: 5′-GATGACGTGGCAAAGAACAG-3′, R: 5′-TCCTCGGGGTCCTGAGTTAT-3′), fatty acid transport protein 5 (Fatp5; F: 5′-GCACCTTCTGACCCAGTACC-3′, R: 5′-GTAAGCAGCCAAGGAATCCA-3′).
3
First-Strand cDNA Synthesis Protocol
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First strand cDNA was synthesised using qScriptTM cDNA Synthesis Kit (Quanta Biosciences). Further, 1μg total RNA was reverse transcribed at a final 20 μL of reaction volume containing 4 μL qScript Reaction Mix, and 1 μL qScript Reverse Transcriptase using MultiGene thermal cycler (Labnet International). PCR amplification protocol was followed according to the manufacturer’s recommendations.
4
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from cells using a PureLink RNA mini kit (Cat number 12183018A; Ambion, USA) according to manufacturer's recommendations and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized from 1 μg RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA) and a MultiGene thermal cycler (Labnet) according to manufacturer's instructions. Relative mRNA levels were inferred from the cDNAs using Power SYBR Green master mix (Applied Biosystems, UK) and TaqMan Universal master mix II, no UNG (Applied Biosystems, USA), both according to manufacturer's instructions, and a real-time PCR detection system (Applied Biosystems). Relative mRNA levels were normalized to the reference gene GAPDH, and then gene expression quantification was performed using a comparative Ct method, wherein ΔCT is defined as the difference between the target and reference gene CT values. Primers are listed in Supplementary Tables
S7 and
S8 . These primers were either TaqMan primers (Applied Biosystems) or custom primers whose sequences have been published previously.
S
S
5
Quantifying Gene Expression in Adipose Tissues
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Total RNA from iWAT, eWAT and BAT was extracted using TRIzol reagent (Thermo Fisher Scientific, Barcelona, Spain) following the manufacturer’s protocol. RNA was quantified in NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The integrity of the RNA was evaluated by the RNA integrity number (RIN) trough 2100 Bioanalyzer Instrument (Agilent Technologies). A RIN higher than six was accepted for total RNA samples. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Barcelona, Spain) in a Multigene ThermalCycler (Labnet, Madrid, Spain). The cDNA was subjected to a quantitative reverse transcriptase polymerase chain reaction amplification using iTaq™Universal SYBR Green Supermix (Bio-Rad, Madrid, Spain) in the 7900HT Fast Real-Time PCR System (Applied Biosystems). The primers used for the different genes are described in Supplementary Table S2 and were obtained from Biomers.net (Ulm, Germany). The relative expression of each gene was calculated according to cyclophilin peptidylprolyl isomerase A (Ppia) mRNA levels and normalized to the levels measured in the corresponding control group. The ∆∆Ct method was used and corrected for primer efficiency [23 ].
6
ILTV p32 Gene Amplification Protocol
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Primers, designed previously by Vögtlin et al. [13 (link)], were used to target a fragment of p32 gene of ILTV. These primers named of ILTU2 5-CTA CGTG CTGGG CTCT AATCC-3 and ILTL2 5-AAACTCT CGGG TGGCT ACTGC-3 produced a 588-bp. The amplification was done by adding 5 µL of the DNA to the PCR mix (Invitrogen™), and the total volume of the reaction was set up to 50 µL. The reaction was carried out in a Multigene Thermal Cycler (Labnet, USA). The denaturation temperature was 95°C for 5 min, then 35 cycles of 1 min at same temperature. The extension temperature was 72°C for 10 min. PCR product was analyzed by electrophoresis in 1.5% agarose gel and visualized under UV light for the 588-bp fragment as shown in Figure-1 .
7
ISSR-based Genomic DNA Amplification
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Ten ISSR primers (Table 2) were used in the present experiment, to amplify the genomic DNA samples. Amplification reactions were carried out in 20μl volumes, containing (4 µl of DNA template (25 ng/ µl), 2 µl of primer (10 pmol) and 10 µl of master mix taq. DNA polymerase, Sigma Scientific Services Co., Egypt). The reaction volume was completed to 20 µl with sterilized deionized water. The Thermal cycler was programmed for initial denaturation at 94 ºC 5 min, 1 min denaturation at 94 ºC, 1 min annealing at 40 ºC and 2 min extension at 72 ºC followed by final extension for 7 min at 72 ºC. DNA denaturation, annealing temperature and number of cycles of each RAPD and ISSR primers are shown in Table (1 and2).
The amplification of RAPD and ISSR primers were carried out in Multigene Thermal Cycler (Labnet). PCR products were resolved by gel electrophoresis on 2% agarose gels in Tris-acetate EDTA (TAE) buffer at 100 volts. Subsequently, gels were stained with Ethidium bromide (0.1g in 10 ml 1X TAE buffer) for 30 min., visualized on UV light and photo-documentation was performed. The size of amplified fragments was estimated according to the standared ladder of 100 bp.
The amplification of RAPD and ISSR primers were carried out in Multigene Thermal Cycler (Labnet). PCR products were resolved by gel electrophoresis on 2% agarose gels in Tris-acetate EDTA (TAE) buffer at 100 volts. Subsequently, gels were stained with Ethidium bromide (0.1g in 10 ml 1X TAE buffer) for 30 min., visualized on UV light and photo-documentation was performed. The size of amplified fragments was estimated according to the standared ladder of 100 bp.
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