To create the cDNA, a 20 μL reverse transcriptase (RT) reaction was completed in a BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA). The first step consisted of 1 μg of total RNA template, 10 μM of random hexamer primers, and 2 mM of oligo-dT primers. The RT protocol was to anneal primers to RNA at 94 °C for 5 min, copy the first strand for 60 min at 42 °C (optimum temperature for the enzyme), then heat inactivate at 70 °C for 15 min and hold at 4 °C until ready to analyze by Nanodrop (Waltham, MA, USA). The concentration of cDNA obtained was determined by measuring the absorbance at 260 nm and 280 nm using an extinction coefficient of 33 (for single stranded DNA). Genomic DNA contamination was assessed by a real-time RT-PCR assay for the reference genes samples.
C1000 touch thermocycler
Manufactured by Promega
Sourced in United States
The C1000 Touch Thermocycler is a versatile and compact instrument designed for precise temperature control during various molecular biology applications. It features a large, intuitive touch screen interface and advanced temperature ramping capabilities to ensure accurate and reproducible results.
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10 protocols using c1000 touch thermocycler
1
Reverse Transcription of Total RNA
2
Duodenal Gene Expression Analysis
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To obtain the cDNA, a total of 20 µL reverse transcriptase (RT) reaction was carried out in a BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA). The concentration of the resulting cDNA was determined by measuring the absorbance at 260 nm and 280 nm, applying an extinction coefficient of 33 (for single-stranded DNA).
For gene expression analysis of the duodenum, a real-time polymerase chain reaction (RT-PCR) was conducted. The primers used in the real-time qPCR were designed based on gene sequences sourced from the Genbank database, utilizing the Real-Time Primer Design Tool software (https://www.idtdna.com/scitools/Applications/RealTimePCR/default.aspx ) (IDT DNA, Coralvilla, IA, USA) [29 (link)]. The sequences and descriptions of the primers used can be found in Table 1 . To assess primer specificity, a BLAST search against the genomic National Center for Biotechnology Information (NCBI) database was performed. The primer for Gallus gallus 18S rRNA was designed as a reference gene.
For gene expression analysis of the duodenum, a real-time polymerase chain reaction (RT-PCR) was conducted. The primers used in the real-time qPCR were designed based on gene sequences sourced from the Genbank database, utilizing the Real-Time Primer Design Tool software (
3
RT-PCR Methodology for Gene Expression
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RT-PCR was conducted as described [24 (link),25 (link),26 (link)]. The cDNA was created from a 20-µL reverse transcriptase (RT) reaction (BioRad C1000 Touch Thermocycler) and using the Improm-II Reverse Transcriptase Kit (Promega, Madison, WI, USA). The cDNA content was assessed by absorbance at 260 and 280 nm, with an extinction coefficient of 33 (single-stranded DNA). Genomic DNA contamination was evaluated via a real-time RT-PCR assay for the reference gene samples [17 (link),18 (link),30 (link)].
The primers used in the real-time PCR were formulated in accordance with relevant gene sequences (GenBank database), via Real-Time Primer Design Tool software (IDT DNA, Coralville, IA, USA) and as was previously described [24 (link),25 (link),26 (link)]. The primer sequences related to iron, zinc, calcium and magnesium metabolism, immune response, hypertension and BBM functionality that were utilized are indicated inTable 1 . The specificity of the primers was verified via BLAST search versus the genomic National Center for Biotechnology Information (NCBI) database. The reference gene used was the 18S rRNA specific for the Gallus gallus model.
The primers used in the real-time PCR were formulated in accordance with relevant gene sequences (GenBank database), via Real-Time Primer Design Tool software (IDT DNA, Coralville, IA, USA) and as was previously described [24 (link),25 (link),26 (link)]. The primer sequences related to iron, zinc, calcium and magnesium metabolism, immune response, hypertension and BBM functionality that were utilized are indicated in
4
cDNA Synthesis and Quantification
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To create the cDNA, a 20 µL reverse transcriptase (RT) reaction was completed in a BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA). The concentration of cDNA obtained was determined by measuring the absorbance at 260 and 280 nm using an extinction coefficient of 33 (for single stranded DNA). Genomic DNA contamination was assessed by a real-time RT-PCR assay for the reference gene samples [12 (link)].
5
RT-PCR Methodology for cDNA Synthesis
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RT-PCR was performed as previously published [23 (link)]. Briefly, in order to create the cDNA, a 20 µL reverse transcriptase (RT) reaction was completed in a BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA). The concentration of cDNA obtained was determined by measuring the absorbance at 260 nm and 280 nm using an extinction coefficient of 33 (for single stranded DNA). Genomic DNA contamination was assessed using a real-time RT-PCR assay for the reference of genes samples. The complete methodology is indicated in the supplementary materials .
6
Reverse Transcription and cDNA Quantification
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From the extracted RNA, cDNA was created by a 20 μL reverse transcriptase (RT) reaction using the BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA). Firstly, 1 μg of total RNA template, 2 mM of oligo-dT primers, and 10 μM of random hexamer primers were added to the given vial. The optimum annealing temperature was 94 °C for 5 min, copying was 60 min at 42 °C followed by heat inactivation at 70 °C for 15 min. The cDNA obtained was analyzed by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) or stored at −80 °C until analysis. The concentration of cDNA was determined by measuring the absorbance at 260 nm and 280 nm with an extinction coefficient of 33 (for single stranded DNA). The extent of genomic DNA contamination was estimated by a RT-PCR assay (real-time) for the reference genes samples.
7
Reverse Transcription for cDNA Synthesis
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To create the cDNA, a 20 µL reverse transcriptase (RT) reaction was completed in a BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega Corp., Madison, WI, USA). The first step consisted of 1 µg of total RNA template, 10 µM of random hexamer primers, and 2 mM of oligo-dT primers. The RT protocol was used to anneal primers to RNA at 94 °C for 5 min. The first strand was copied for 60 min at 42 °C (optimum temperature for the enzyme), followed by exposure to 70 °C (15 min) for enzymatic inactivation, samples were then held at 4 °C until quantification by a Nanodrop™ ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). The concentration of cDNA obtained was determined by measuring the absorbance at 260 nm and 280 nm using an extinction coefficient of 33 (for single stranded DNA). Genomic DNA contamination was assessed by a real-time RT-PCR assay for the reference gene samples.
8
cDNA Synthesis and Quantification
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cDNA was created using the 20 µL reverse transcriptase (RT) reaction, performed in a BioRad C1000 Touch Thermocycler using the Improm-II Reverse Transcriptase Kit (Promega, Madison, WI, USA). The concentration of cDNA was assessed using Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). Further details can be found in a previous publication [31 (link)].
9
cDNA Synthesis Using Improm-II Kit
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The complementary DNA (cDNA) reaction was conducted as previously described (BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit, Promega Corp., Madison, WI, USA) [37 (link),38 (link),39 (link)].
10
RT-PCR Quantification of Gene Expression
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RT-PCR was performed as previously published [39, 42, 43] . Briefly, 20 µL reverse transcriptase (RT) reaction was completed in a BioRad C1000 Touch Thermocycler applying the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA) to form the cDNA. cDNA concentration was quantified by the absorbance at 260/280 nm using an extinction coefficient of 33 (for singlestranded DNA). Genomic DNA contamination was measured by a real-time RT-PCR assay for the reference gene samples [44] [45] [46] .
The primers used in the real-time PCR were designed. This procedure was based on gene sequences from the GenBank database, using Real-Time Primer Design Tool software (IDT DNA, Coralville, IA, USA), as previously described [39, 42, 43] . Primers sequences used in this study were summarized in Table 1. Through performing a BLAST search against the genomic National Center for Biotechnology Information (NCBI) database, the specificity of the primers was tested. The reference gene used was the 18S rRNA specific for the Gallus gallus model.
The primers used in the real-time PCR were designed. This procedure was based on gene sequences from the GenBank database, using Real-Time Primer Design Tool software (IDT DNA, Coralville, IA, USA), as previously described [39, 42, 43] . Primers sequences used in this study were summarized in Table 1. Through performing a BLAST search against the genomic National Center for Biotechnology Information (NCBI) database, the specificity of the primers was tested. The reference gene used was the 18S rRNA specific for the Gallus gallus model.
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