Sixteen loci on SSC7 were selected on the basis of the GWAS results, 3 of which were related to BF, while the others were related to TN. Primers for genotyping were designed and ordered on the Fluidigm D3 assay design website (Supplementary Table S13 ), and 191 out of the total 2,869 pigs were genotyped for each SNP using Fluidigm Dynamic array IFC.
Dynamic array ifc
Manufactured by Standard BioTools
The Dynamic Array IFC is a microfluidic integrated circuit (IFC) designed for high-throughput gene expression analysis. It enables the simultaneous measurement of multiple target genes across a large number of samples using the Fluidigm technology platform. The core function of the Dynamic Array IFC is to facilitate precise and efficient gene expression profiling.
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Lab products found in correlation
Evagreen supermix, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Prism 6, by GraphPad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Fluidigm biomark hd system, by Standard BioTools (1 mentions)
Biomark hd system, by Standard BioTools (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using dynamic array ifc
1
SNP Genotyping on Pig Trait Loci
2
Fluidigm Array Data Validation
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For validation of array data by Fluidigm technology 22 animals (88 samples) were kept to fit the technical constraints of this technique. Total RNA (1 μg) used in microarray experiments was reverse-transcribed as previously described [15 (link)]. Primer sequences for genes were designed using Primer3plus software (http://primer3plus.com ) and are given in Additional file 1 . The TFRC gene (transferrin receptor) and EPRS gene (glutamyl-prolyl-tRNA synthetase) were used as internal controls. Pre-amplified samples were analyzed with a 96 ×96 Dynamic Array™ IFC (Fluidigm) following the protocol defined by [16 (link)], with some modifications. All measurements were performed on the same plate. Each gene was tested twice for each sample. Four dilution points containing a pool of all samples were used to determine PCR efficiency. Data were analyzed using BioMark Gene Expression Data Analysis software (Fluidigm) to obtain Ct values. The Pfaffl method was applied to compute the relative expression of each gene [17 (link)]. Pearson correlations were computed to compare the expression values of microarray and quantitative real-time PCR. Quantitative RT-PCR data were also analyzed for time effect by ANOVA with repeated measurements for every gene.
3
Chicken Skin Developmental Transcriptome
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Skin samples were collected from generation F8 of the HB × HQLA intercross including embryos at embryonic day (E) 7.5, 8.5, 9.5 and 10.5, chicks of two-week-old and adult chickens. RNAs derived from embryo tissue were isolated using RNeasy micro kit (Qiagen), while RNAs from other tissue were homogenized in TRIzol (Invitrogen) followed by DNase I treatment and clean-up using the RNeasy mini kit (Qiagen). cDNAs were generated with 1 μg RNA using Reverse Transcription System (Promega) in a 20 μl volume. The cDNAs were then applied to specific target amplifications, respectively, using a forward and reverse primer mix with each primer at a concentration of 100 μM in a 5 μl volume to increase the number of copies of target genes. Then a cleanup step with Exonuclease I was performed to remove unincorporated primers, and the final products were diluted before qPCR reactions. After loading samples and assays into the Dynamic Array IFC (Fluidigm), cDNA levels were quantified by qPCR using SsoFast EvaGreen Supermix (Bio-Rad) with Fluidigm Biomark HD system. Samples were run in sextuplicate using EvaGreen Supermix (Bio-rad) and normalized to GAPDH. The 2-ΔΔCT method was used to analyze the relative changes in gene expression. Statistical analysis was performed with GraphPad Prism 6 (GraphPad Software, San Diego, CA).
4
Fluidigm PCR for Gene Expression
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The primers used in this study as well as reaction efficiencies are listed in our previous paper [28 ]. PCR-primer pairs were designed from mRNA sequences of the studied genes provided by the National Center for Biotechnology Information (NCBI) gene database using PrimerBlast. Results were retained if the reaction efficiency was between 80% and 110%. The optimal Tm at 60°C and exon junction span were selected. The primers were ordered at 100 µM concentration from Eurofins Genomics, Ebersberg, Germany.
One microgram of extracted RNA was retrotranscribed using the SuperScriptTM VILOTM cDNA synthesis kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. For Fluidigm PCR, cDNA was preamplified according to Fluidigm’s protocol (quick reference PN 100–5875 B1). Preamplified cDNA was then diluted fivefold with Tris-EDTA buffer. Gene expression levels were measured on 48 × 48 GE Dynamic Array IFC using the Fluidigm BioMarkTM HD System. Fold changes were calculated by the ∆∆Ct method using ‘Fluidigm Real-Time PCR Analysis’ software. The reference genes used were GAPDH, CPR2, and HPRT1 [30 (link)]. Each expression value was normalized to one reference sample – mammary tissue from the first lactation with inflammation. This allowed for a more reliable reference for genes whose expression is activated by inflammation.
One microgram of extracted RNA was retrotranscribed using the SuperScriptTM VILOTM cDNA synthesis kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. For Fluidigm PCR, cDNA was preamplified according to Fluidigm’s protocol (quick reference PN 100–5875 B1). Preamplified cDNA was then diluted fivefold with Tris-EDTA buffer. Gene expression levels were measured on 48 × 48 GE Dynamic Array IFC using the Fluidigm BioMarkTM HD System. Fold changes were calculated by the ∆∆Ct method using ‘Fluidigm Real-Time PCR Analysis’ software. The reference genes used were GAPDH, CPR2, and HPRT1 [30 (link)]. Each expression value was normalized to one reference sample – mammary tissue from the first lactation with inflammation. This allowed for a more reliable reference for genes whose expression is activated by inflammation.
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