Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data

Jureckova, Katerina; Raschmanová, Hana; Kolek, Jan; Vasylkivska, Maryna; Branská, Barbora; Patáková, Petra; Provazník, Ivo; Sedlář, Karel

doi:10.3389/fmicb.2021.640054

Cited by 4 publications

(3 citation statements)

References 57 publications

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“…The core genome can find additional utilization in the identification of versatile reference genes for RT-qPCR in C. beijerinckii, as such genes should be present in every strain. However, experimental validation is always needed, as a reference gene-coding peptidase T (Gene4499# in the core genome), proposed as a reference gene for the strain NCIMB 8052 [20], was later proved to be not utilizable for the strain NRRL B-598 [78], where genes greA (Gene2564# in the core genome), zmp (Gene1191# in the core genome) and others performed better.…”

Section: Discussionmentioning

confidence: 99%

Diversity and Evolution of Clostridium beijerinckii and Complete Genome of the Type Strain DSM 791T

et al. 2021

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Clostridium beijerinckii is a relatively widely studied, yet non-model, bacterium. While 246 genome assemblies of its various strains are available currently, the diversity of the whole species has not been studied, and it has only been analyzed in part for a missing genome of the type strain. Here, we sequenced and assembled the complete genome of the type strain Clostridium beijerinckii DSM 791T, composed of a circular chromosome and a circular megaplasmid, and used it for a comparison with other genomes to evaluate diversity and capture the evolution of the whole species. We found that strains WB53 and HUN142 were misidentified and did not belong to the Clostridium beijerinckii species. Additionally, we filtered possibly misassembled genomes, and we used the remaining 237 high-quality genomes to define the pangenome of the whole species. By its functional annotation, we showed that the core genome contains genes responsible for basic metabolism, while the accessory genome has genes affecting final phenotype that may vary among different strains. We used the core genome to reconstruct the phylogeny of the species and showed its great diversity, which complicates the identification of particular strains, yet hides possibilities to reveal hitherto unreported phenotypic features and processes utilizable in biotechnology.

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Section: Discussionmentioning

confidence: 99%

Diversity and Evolution of Clostridium beijerinckii and Complete Genome of the Type Strain DSM 791T

et al. 2021

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“…Since the number of RNAs that are analyzed in the validation cohort has been reduced to a smaller subset of biomarker candidates, a more time- and cost-efficient method can be used. RT-qPCR is a well-established and—even more relevant—standardized method to confirm and validate the candidate biomarker signatures when in compliance with the MIQE guidelines, as was shown by several recently published studies [ 20 , 86 , 87 , 88 ].…”

Section: From Biomarker Development To Routine Diagnosticsmentioning

confidence: 99%

Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery

Grätz

Bui

Thaqi

et al. 2022

Life

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In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of “key steps” in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.

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“…Therefore, a systematic research on stably expressed RGs of A. acidoterrestris under stress conditions is required. The RNA-seq technique provides not only a method to determine gene expression at the transcriptome level but also a novel approach for RGs prediction, which has been successfully applied in multiple species (Alexander et al, 2012;He et al, 2019;Zhang et al, 2020;Jureckova et al, 2021). Therefore, it was hypothesized that the novel and reliable RGs for investigating stress responses in A. acidoterrestris could be predicted using transcriptome data.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress

Zhao

Jiao

et al. 2021

Front. Microbiol.

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Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

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Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data

Cited by 4 publications

References 57 publications

Diversity and Evolution of Clostridium beijerinckii and Complete Genome of the Type Strain DSM 791T

Diversity and Evolution of Clostridium beijerinckii and Complete Genome of the Type Strain DSM 791T

Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress

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