RNA was isolated from chicken tissues and skin models28 (link) and purified with TriFast according to a published protocol46 (link) and reverse-transcribed with the iScriptTM cDNA synthesis kit (Biorad, Hercules, CA). Polymerase chain reactions (PCRs) were performed with primer pairs specific for KRT9LC2 (KRT9LC2-s, 5′-GAATGCCGCTACAACAACCAC-3′ and KRT9LC2-a, 5′-TGCTTCAGGGATCTCTCATTG-3′), IL1RN (IL1RN-s, 5′-GAGAAGGTGTTTTGGGTGCC-3′ and IL1RN-a, 5′-TAGGTGCGGAAGAAGGTGAA-3′), IL36RN (IL36RN-s, 5′-GAGCTCAGCCGTACCACTAC-3′ and IL36RN-a, 5′-AACAGCTTCACCTCCTCCAG-3′), and the housekeeping gene Hydroxymethylbilane synthase (HMBS) (HMBS-s, 5′-AACTGTGGGAAAACGCTCAG-3′ and HMBS-a, 5′-TTCTCTTCAGTCCAGCAGCA-3′) on a Roche LightCycler with LC480 SYBR Green I Master Kit according to the manufacturer’s protocol. Quantitative analysis of IL1RN and IL36RN expression in chicken tissues was performed according to a published method46 (link). The expression levels of these genes were compared between scutate scales and other tissues, considering differences with a P value of < 0.05 significant (two-sided t-test).
Lc480 sybr green 1 master kit
Manufactured by Roche
Sourced in New Zealand
The LC480 SYBR Green I Master Kit is a laboratory equipment product from Roche. It is a reagent kit designed for real-time PCR analysis using the SYBR Green I dye for gene expression studies and quantification. The kit includes the necessary components for performing real-time PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 system, by Roche (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Lightcycler, by Roche (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Revertaid h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Lightcycler 480ii, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Dnase 1, by Promega (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Lc 480 sybr green 1 master mix, by Roche (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Bioruptor next gen, by Diagenode (1 mentions)
6 protocols using lc480 sybr green 1 master kit
1
Quantitative Analysis of Chicken Tissue Gene Expression
2
Quantitative Real-Time PCR Analysis
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Total RNA was extracted with the Spectrum Plant total RNA kit (Sigma). cDNA synthesis was performed using these RNA preparations, oligo-dT primers, the Ribolock RNAse inhibitor and RevertAid H Minus reverse transcriptase (all reagents from Thermo-Fisher). Relative transcript abundance of selected genes (see Supplementary Table 3 for a list of genes and the primers used) was determined using the Roche LightCycler 480 system and the LC480 SYBR Green I Master kit (Roche Applied Sciences). LightCycler melting curves were obtained for the reactions, revealing single peak melting curves for all amplification products. The amplification data were analyzed using the second derivative maximum method, and resulting Cp values were converted into relative expression values using the comparative Ct method44 (link). For each sample, one reference gene was used to normalize the data: ‘REF2’ (At4g34270) for the hormone treatments, ‘REF1’ (At1g13320) to monitor gene expression of defense-related genes following Pst AvrRpm1 or mock-inoculated tissue45 (link), and MON1 (At2g28390) to check expression of N-end rule related genes after inoculation with Pst AvrRpm1.
3
Genomic DNA Extraction and 16S rRNA Amplification
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Genomic DNA was extracted from 200 μL of EMJH liquid clonal cultures. Extraction was performed using a commercial kit (QIAamp® DNA Mini Kit, Qiagen, Australia) according to the manufacturer's instructions. DNA elution was performed with 50 μL of buffer AE. The quantity of DNA was measured with NanoDrop™ (Thermo Fisher Scientific, Waltham, MA USA) and adjusted to 0.1 ng/μL.
Amplification of a fragment of the 16S rRNA gene was performed in a total volume of 10 μL on a LightCycler 480 II. The reaction mixture consisted in 2 μL PCR grade water, 5 μL of 2X reaction SYBR Green Mix (LC480 SYBR Green I Master kit, Roche, New Zealand), 0.5 μL of forward primer (5′-GGCGGCGCGTCTTAAACATG-3′), 0.5 μL of reverse primer (5′-CTTAACTGCTGCCTCCCGTA-3′), and 2 μL (0.2 ng) of DNA (Mérien et al., 1992 (link)). After amplification, the melting temperature of the amplified product was analyzed using LightCycler 480 Software.
Amplification of a fragment of the 16S rRNA gene was performed in a total volume of 10 μL on a LightCycler 480 II. The reaction mixture consisted in 2 μL PCR grade water, 5 μL of 2X reaction SYBR Green Mix (LC480 SYBR Green I Master kit, Roche, New Zealand), 0.5 μL of forward primer (5′-GGCGGCGCGTCTTAAACATG-3′), 0.5 μL of reverse primer (5′-CTTAACTGCTGCCTCCCGTA-3′), and 2 μL (0.2 ng) of DNA (Mérien et al., 1992 (link)). After amplification, the melting temperature of the amplified product was analyzed using LightCycler 480 Software.
4
Quantitative Gene Expression Analysis
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RNA was extracted using the RNeasy Plant Mini Kit (Qiagen), and treated with DNase I (Promega) prior to complementary DNA (cDNA) synthesis with the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions. Relative transcript abundance of selected genes (for a list of genes and the primers used, see Supplementary Table 4 ) was determined using the Roche LightCycler 480 system and the LC480 SYBR Green I Master Kit (Roche Diagnostics). Measurements were taken for three technical repeats. The amplification data were analysed using the second derivative maximum method, and resulting cycle threshold values were converted into relative expression values using the comparative cycle threshold method.
5
Quantification of Bile Acid Receptor in Rat Colon
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G protein-coupled bile acid receptor 1 (GpBAR1) is the bile acid receptor that is expressed in rat colonic epithelium, enterochromaffin cells, and myenteric neurons in the colon.14 (link),16 (link) The polymerase chain reaction (PCR) amplification was performed in a LightCycler 480 Real-Time PCR System (Roche Applied Science, Indianapolis, IN, USA) in 96 well plates. PCR mix contained 2 μL cDNA template, 2 μL of primer mix and 10 μL of LC480 SYBR Green I Master Mix from the LC480 SYBR Green I Master kit (Roche). Plate layout design was maintained for triplicates of the samples according to rat GpBAR1 (forward 5′-cactgcccttctctctgtcc-3′; reverse 5′-agttcaggtccagttacgc-3′) and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward 5′-ggtgctgagtatgtcgtgga-3′; reverse 5′-gtggttcacacccatcacaa-3′), 3 wells for negative. The PCR protocol included 1 cycle of polymerase activation for 10 minutes at 95°C, 45 cycles of amplification with 10 seconds denaturation at 95°C, 1 minute annealing at 55°C, and 5 seconds extension at 72°C in each cycle and 1 cycle for melting curve analysis with 10 seconds denaturation at 95°C, 1 minute annealing at 60°C and a melting step at 98°C at 0.2°C/sec ramp rate. Each sample was run in triplicate.
6
Chromatin Immunoprecipitation of FRS12 in Arabidopsis
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Seedlings expressing ProFRS12:FRS12-HA and Col-0 wt were grown in MS-agar medium for 10 days, harvested in liquid nitrogen at the indicated time points and stored at −80 °C before analysis. Plant material was grinded, lysed and crosslinked using formaldehyde. The crosslinking reaction was stopped using glycine. Nuclei were isolated and lysed in a sucrose gradient and the chromatin obtained was fragmented by sonication (Bioruptor Next Gen, Diagenode). Immunoprecipitations were performed using anti-HA (3F10 Roche)-coated IgG magnetic beads (Dynabeads protein G 1003D, Invitrogen). Protein–DNA complexes were eluted using 0.1M NaHCO3 and 1% w/w SDS. Reverse DNA crosslinking was performed in two steps: overnight at 65 °C using 0.2 M NaCl and 1 h incubation at 45 °C adding 10 μl 0.5 M EDTA, 20 μl 1 M Tris HCl pH 6.5 and 2 μl 10 mg ml−1 proteinase K. DNA was extracted using phenol-chloroform isoamyl alcohol pH8 and precipitated with sodium acetate/glycogen/ethanol63 (link). The qPCR analysis was performed with 0.5 μl of sample per reaction using and the LC480 SYBR Green I Master Kit (Roche Diagnostics) on a Roche LightCycler 480 system. For each pair of primers, normalization to DNA input was carried out. The fold enrichment was calculated as relative to Col-0 wt using the ΔCt expression values.
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