Total RNA was extracted from the scaffold using Trizol reagent (Invitrogen™, Waltham, MA, USA). Each cDNA sample was tested in triplicate. Quantitative RT-PCR analysis was performed in LineGene 9640 Bioer (CaRli biotec S.r.l, Roma, Italy) using SYBR® Green Real-Time PCR Master Mixes (Applied Bio-systems™, Life Technologies—EuroClone S.p.A, Pero, Milan, Italy). QuantiTect Primers (Qiagen Srl, Milan, Italy) and designed primers (Invitrogen™) were used (Table S2 ). The expression of target genes was analyzed performing the 2−ΔΔCT method, where the β-actin expression was used as the reference gene. The results obtained are expressed as the “relative fold” of changes calculated with respect to untreated cells; these data were used as a calibrator for each experimental point. All procedures have been performed in accordance with the manufacturer’s instructions.
Designed primers
Manufactured by Thermo Fisher Scientific
Sourced in Italy, United States
Designed primers are short, synthetic DNA sequences used as starting points for DNA amplification in various molecular biology techniques, such as polymerase chain reaction (PCR). They are designed to target specific regions of the DNA, allowing for the selective and controlled replication of targeted genetic sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green real time pcr master mixes, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Primer express 3, by Thermo Fisher Scientific (1 mentions)
4 protocols using designed primers
1
Quantitative Analysis of Gene Expression
2
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated from arcuate nucleus samples with QIAGEN RNeasyMiniKit (Qiagen, Valencia, CA, USA) according the manufacturer’s instruction. To eliminate genomic DNA contamination, DNase I (Fermentas) treatment was used. Sample quality control and the quantitative analysis were carried out by NanoDrop (Thermo Scientific). Amplification was not detected in the RT-minus controls. cDNA synthesis was performed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Designed primers (Invitrogen) were used in real-time PCR reaction with Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) on ABI StepOnePlus instrument. The amplicon was tested by Melt Curve Analysis and mRNA levels were calculated by ABI StepOne 2.3 program using ΔΔCT method. Experiments were normalized to GAPDH expression. Primer sequences are listed in Supplemental Material.
3
Quantitative RT-PCR Analysis of Scaffold RNA
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Total RNA was extracted from the scaffold using Trizol reagent (Invitrogen™).
Each cDNA sample was tested in duplicate. Quantitative RT-PCR analysis was
performed in a LightCycler 2.0 Instrument (Roche Diagnostics SpA, Milan, Italy)
using SYBR® Green Real-Time PCR Master Mixes (Applied Biosystems™, Life
Technologies - EuroClone S.p.A). QuantiTect Primers (Qiagen Srl, Milan, Italy)
and designed primers (Invitrogen™) were used (Tab. I ). Gene expression analysis was
performed employing the 2−ΔΔCT method using GAPDHexpression as reference gene (48 (link)). Results were expressed as relative fold changes calculated
using Untreated Scaffold data as calibrator for each experimental time
point.
Each cDNA sample was tested in duplicate. Quantitative RT-PCR analysis was
performed in a LightCycler 2.0 Instrument (Roche Diagnostics SpA, Milan, Italy)
using SYBR® Green Real-Time PCR Master Mixes (Applied Biosystems™, Life
Technologies - EuroClone S.p.A). QuantiTect Primers (Qiagen Srl, Milan, Italy)
and designed primers (Invitrogen™) were used (
performed employing the 2−ΔΔCT method using GAPDHexpression as reference gene (48 (link)). Results were expressed as relative fold changes calculated
using Untreated Scaffold data as calibrator for each experimental time
point.
4
Quantitative Real-Time PCR Measurement Protocol
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For quantitative real time PCR measurements, tissue samples were cut and immediately frozen in dry ice. Total RNA was isolated from colonic and cell culture samples with QIAGEN RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according the manufacturer’s instruction. To eliminate genomic DNA contamination DNase I treatment was used (100 μl RNase-free DNase I (1 U DNase I, Fermentas) solution was added). Sample quality control and the quantitative analysis were carried out by NanoDrop (Thermo Scientific). Amplification was not detected in the reverse transcription (RT)-minus controls. cDNA synthesis was performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The designed primers (Invitrogen) were used in the Real-Time PCR reaction with Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) on ABI StepOnePlus instrument. The gene expression was analyzed by ABI StepOne 2.3 program. The amplicon was tested by Melt Curve Analysis on ABI StepOnePlus instrument. GAPDH was used as endogenous control reference gene and all data were normalized to GAPDH expression. Primers used for the comparative CT experiments were designed by the Primer Express 3.0 program (Applied Biosystems). Primer sequences are given in the supplementary material .
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