Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris).
Housekeeping genes
Komagataella phaffii
Pichia pastoris
RT-qPCR
Relative transcript analysis
Transcriptomics
Journal
Fungal biology and biotechnology
ISSN: 2054-3085
Titre abrégé: Fungal Biol Biotechnol
Pays: England
ID NLM: 101655873
Informations de publication
Date de publication:
29 Mar 2023
29 Mar 2023
Historique:
received:
14
02
2023
accepted:
10
03
2023
medline:
30
3
2023
entrez:
29
3
2023
pubmed:
30
3
2023
Statut:
epublish
Résumé
The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.
Sections du résumé
BACKGROUND
BACKGROUND
The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools.
RESULTS
RESULTS
We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays.
CONCLUSION
CONCLUSIONS
The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.
Identifiants
pubmed: 36991508
doi: 10.1186/s40694-023-00154-1
pii: 10.1186/s40694-023-00154-1
pmc: PMC10061771
doi:
Types de publication
Journal Article
Langues
eng
Pagination
7Subventions
Organisme : Austrian Research Promotion Agency
ID : 880555
Organisme : Austrian Science Fund,Austria
ID : P 35642
Informations de copyright
© 2023. The Author(s).
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