Inflamed pinch biopsies from 6 CD patients and normal biopsies from 6 healthy controls were sent in RNA stabilization solution RNAlater® (Ambion, Life Technologies, Carlsbad, CA) to AROSAB (Aarhus, Denmark) for mRNA analysis. RNA was extracted from the biopsy tissue that had been cultured overnight to quantify cytokine release in the explant assay. An Applied Biosystems 7900HT platform was used to perform qPCR experiments with TaqMan reactions (Thermo Fisher Scientific, Waltham, MA). Transcripts were analyzed with TaqMan probes for KLRK1 (NKG2D), MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
Taqman reactions
Manufactured by Thermo Fisher Scientific
TaqMan reactions are a widely used technique in molecular biology and diagnostics. They utilize fluorogenic probe-based chemistry to enable real-time detection and quantification of DNA or RNA targets during the polymerase chain reaction (PCR) process. This technology provides a reliable and sensitive method for analyzing gene expression, detecting pathogens, and quantifying nucleic acid sequences.
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Lab products found in correlation
2 protocols using taqman reactions
1
Comparative Analysis of NKG2D Ligands in CD
2
Quantitative RNA Expression Analysis
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Total RNA of cell-cultured TCs was isolated using the GenElute Mammalian Total RNA Purification Kit (Merck) according to the manufacturer’s instructions. First, 1,000 ng of RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. Gene expression analysis was performed by quantitative PCR using TaqMan reactions (Thermo Fisher Scientific) (Supplementary Materials Table) and Lightcycler 480 (Roche). Gene expression levels were assessed using the Ct method and normalized to the expression of Actb, resulting in ΔCt values. Relative gene expression was assessed by normalization of ΔCt values of individual samples to the average control ΔCt value, resulting in ΔΔCt values. Relative FCs to control were then calculated as 2−ΔΔCt.
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