LC-MS and gene expression.xlsx (3.03 MB)

LC-MS and gene expression.xlsx

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posted on 2022-08-26, 13:44 authored by Luiza Moraes HolstLuiza Moraes Holst, Cristina Iribarren, Maria Sapnara, Stefan Isaksson, Otto Savolainen, Hans Tornblom, Yvonne Wettergren, Hans Strid, Magnus Simren, Maria K. Magnusson, Lena Ohman

Metabolites of the fecal supernatants were analyzed at Chalmers Mass Spectrometry Infrastructure (Gothenburg, Sweden) using a non‐targeted liquid chromatography–mass spectrometry (LC‐MS) approach. Briefly, the analyses were carried out using reversed‐phase chromatography and hydrophilic interaction chromatography with both positive and negative electrospray ionization. The samples of each study group were analyzed in separate batches, which included its own quality control samples. The analytical workflow named “notame”, described by Zheng et al, was used to pre‐process the acquired data and included drift correction within and between batches, data imputation was done using the missForest R package and clustering of features was done to remove weak and repeated features(24). Log10 transformation was applied prior between‐batch‐correction to reduce possible batch effects caused by the instrument

For gene expression data, a custom RT² PCR Array (Qiagen) was analyzed in a Quantstudio 12K Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the RT² qPCR SYBR Green ROX MasterMix (Qiagen). The gene expression custom array comprised 96 genes (supplementary table 1), including 5 housekeeping (HK) genes (GAPDH, HPRT1, RPLP0, ACTB, B2M) and quality 4 controls (positive PCR control, reverse transcription control, genomic DNA control, negative template control). All samples passed the quality controls. Missing values and CT values >38 were set to 38. Genes were excluded from the analysis if >50% of the samples had missing or very high CT values (CT>36). In total, 18 genes were excluded from the Caco-2 cell analysis, and 19 genes were excluded from the colonoid analysis.  Normalized values are expressed as 2-(CT Target gene-mean CT of HK genes)


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