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A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets.
Nevone, Alice; Lattarulo, Francesca; Russo, Monica; Panno, Giada; Milani, Paolo; Basset, Marco; Avanzini, Maria Antonietta; Merlini, Giampaolo; Palladini, Giovanni; Nuvolone, Mario.
Affiliation
  • Nevone A; Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy.
  • Lattarulo F; Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
  • Russo M; Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy.
  • Panno G; Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
  • Milani P; Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy.
  • Basset M; Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
  • Avanzini MA; Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy.
  • Merlini G; Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
  • Palladini G; Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy.
  • Nuvolone M; Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
Biomedicines ; 11(4)2023 Apr 03.
Article in En | MEDLINE | ID: mdl-37189697
In the next-generation sequencing era, RT-qPCR is still widely employed to quantify levels of nucleic acids of interest due to its popularity, versatility, and limited costs. The measurement of transcriptional levels through RT-qPCR critically depends on reference genes used for normalization. Here, we devised a strategy to select appropriate reference genes for a specific clinical/experimental setting based on publicly available transcriptomic datasets and a pipeline for RT-qPCR assay design and validation. As a proof-of-principle, we applied this strategy to identify and validate reference genes for transcriptional studies of bone-marrow plasma cells from patients with AL amyloidosis. We performed a systematic review of published literature to compile a list of 163 candidate reference genes for RT-qPCR experiments employing human samples. Next, we interrogated the Gene Expression Omnibus to assess expression levels of these genes in published transcriptomic studies on bone-marrow plasma cells from patients with different plasma cell dyscrasias and identified the most stably expressed genes as candidate normalizing genes. Experimental validation on bone-marrow plasma cells showed the superiority of candidate reference genes identified through this strategy over commonly employed "housekeeping" genes. The strategy presented here may apply to other clinical and experimental settings for which publicly available transcriptomic datasets are available.
Key words

Full text: 1 Collection: 01-internacional Database: MEDLINE Language: En Journal: Biomedicines Year: 2023 Document type: Article Affiliation country: Italy Country of publication: Switzerland

Full text: 1 Collection: 01-internacional Database: MEDLINE Language: En Journal: Biomedicines Year: 2023 Document type: Article Affiliation country: Italy Country of publication: Switzerland