Dataset Information

Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients.

ABSTRACT:

Objective

To screen and validate reference genes suitable for gene mRNA expression study in peripheral blood mononuclear cells (PBMCs) between septic patients and healthy controls (HC).

Methods

Total RNA in PBMCs was extracted and RT-qPCR was used to determine the mRNA expression profiles of 9 candidate genes, including ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, RPL13A, SDHA and YWHAZ. The genes expression stabilities were assessed by both geNorm and NormFinder software.

Results

YWHAZ was the most stable gene among the 9 candidate genes evaluated by both geNorm and NormFinder in mixed and sepsis groups. The most stable gene combination in mixed group analyzed by geNorm was the combination of GAPDH, PKG1 and YWHAZ, while that in sepsis group was the combination of ACTB, PKG1 and YWHAZ.

Conclusion

Our first systematic analysis of the reference genes in PBMC of septic patients suggested YWHAZ was the best candidate. The combination of ACTB, PKG1 and YWHAZ could improve RT-qPCR accuracy in septic patients. Our results identified the most stable reference genes to standardize RT-qPCR of sepsis patients, which can serve as a useful tool for gene function exploration in the future.

SUBMITTER: Song R

PROVIDER: S-EPMC10119759 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Publications

Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients.

Song Ruoyu R He Shijun S Wu Yongbin Y Chen Wanxin W Song Jie J Zhu Yaxi Y Chen Huan H Wang Qianlu Q Wang Shouman S Tan Sichuang S Tan Sipin S

Heliyon 20230407 4

<h4>Objective</h4>To screen and validate reference genes suitable for gene mRNA expression study in peripheral blood mononuclear cells (PBMCs) between septic patients and healthy controls (HC).<h4>Methods</h4>Total RNA in PBMCs was extracted and RT-qPCR was used to determine the mRNA expression profiles of 9 candidate genes, including ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, RPL13A, SDHA and YWHAZ. The genes expression stabilities were assessed by both geNorm and NormFinder software.<h4>Results</h4> ...[more]

PMID: 37089378

Similar Datasets

Project description:BackgroundA critical step in the RT-qPCR workflow for studying gene expression is data normalization, one of the strategies being the use of reference genes. This study aimed to identify and validate a selection of reference genes for relative quantification in Talaromyces versatilis, a relevant industrial filamentous fungus. Beyond T. versatilis, this study also aimed to propose reference genes that are applicable more widely for RT-qPCR data normalization in filamentous fungi.ResultsA selection of stable, potential reference genes was carried out in silico from RNA-seq based transcriptomic data obtained from T. versatilis. A dozen functionally unrelated candidate genes were analysed by RT-qPCR assays over more than 30 relevant culture conditions. By using geNorm, we showed that most of these candidate genes had stable transcript levels in most of the conditions, from growth environments to conidial germination. The overall robustness of these genes was explored further by showing that any combination of 3 of them led to minimal normalization bias. To extend the relevance of the study beyond T. versatilis, we challenged their stability together with sixteen other classically used genes such as β-tubulin or actin, in a representative sample of about 100 RNA-seq datasets. These datasets were obtained from 18 phylogenetically distant filamentous fungi exposed to prevalent experimental conditions. Although this wide analysis demonstrated that each of the chosen genes exhibited sporadic up- or down-regulation, their hierarchical clustering allowed the identification of a promising group of 6 genes, which presented weak expression changes and no tendency to up- or down-regulation over the whole set of conditions. This group included ubcB, sac7, fis1 and sarA genes, as well as TFC1 and UBC6 that were previously validated for their use in S. cerevisiae.ConclusionsWe propose a set of 6 genes that can be used as reference genes in RT-qPCR data normalization in any field of fungal biology. However, we recommend that the uniform transcription of these genes is tested by systematic experimental validation and to use the geometric averaging of at least 3 of the best ones. This will minimize the bias in normalization and will support trustworthy biological conclusions.

Dataset Information

Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients.

Objective

Methods

Results

Conclusion

Publications

Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients.

Similar Datasets

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