Quantitative Real-Time PCR Protocol

The lung and intestinal samples were stored in RNAlater (Ambion, Ghent, Belgium) and RNA was extracted using the Aurum kit (Bio-Rad, Nazareth-Eke, Belgium). cDNA was synthesized using the iScript cDNA synthesis Kit (Bio-Rad) and real time qPCR was performed on the CFX384 detection system (Bio-Rad), using the SensiMix SYBR No-ROX mix (Bioline, Kampenhout, Belgium). The expression levels were normalized to the most stable housekeeping genes, which were determined for each organ using the geNorm Housekeeping Gene Selection Software within qBASE+ (Biogazelle, Zwijnaarde, Belgium) [41 (link)]. The primer sequences are listed in Table 5.

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Goossens E., Li J., Callens C., Van Rysselberghe N., Kettunen H., Vuorenmaa J., Garcia Gonzalez N., Libert C., Ducatelle R, & Van Immerseel F. (2022). Acute Endotoxemia-Induced Respiratory and Intestinal Dysbiosis. International Journal of Molecular Sciences, 23(19), 11602.

Publication 2022

RNAlater Aurum kit iScript cDNA synthesis Kit CFX384 detection system SensiMix SYBR No-ROX mix qBASE+

Cdna Housekeeping gene Intestinal Lung Primer Synthesis

Corresponding Organization : Ghent University

Other organizations : Shanxi Agricultural University, Outokumpu (Finland), Vlaams Instituut voor Biotechnologie

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of genes (not specified)

control variables

Storage of lung and intestinal samples in RNAlater
RNA extraction using the Aurum kit
CDNA synthesis using the iScript cDNA synthesis Kit
Real-time qPCR using the CFX384 detection system and the SensiMix SYBR No-ROX mix
Normalization of expression levels to the most stable housekeeping genes, determined using the geNorm Housekeeping Gene Selection Software within qBASE+

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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