Selection and Validation of Reference Genes for Pan-Cancer in Platelets Based on RNA-Sequence Data

Xiaoxia Wen; Guishu Yang; Yongcheng Dong; Liping Luo; Bangrong Cao; Birga Anteneh Mengesha; Ruiling Zu; Yulin Liao; Chang Liu; Shi Li; Yao Deng; Kaijiong Zhang; Xin Ma; Jian Huang; Dongsheng Wang; Keyan Zhao; Ping Leng; Huaichao Luo

doi:10.3389/fgene.2022.913886

Selection and Validation of Reference Genes for Pan-Cancer in Platelets Based on RNA-Sequence Data

Front Genet. 2022 Jun 13:13:913886. doi: 10.3389/fgene.2022.913886. eCollection 2022.

Authors

Xiaoxia Wen¹, Guishu Yang², Yongcheng Dong³, Liping Luo⁴, Bangrong Cao⁴, Birga Anteneh Mengesha⁵, Ruiling Zu⁶, Yulin Liao⁶, Chang Liu⁶, Shi Li⁶, Yao Deng⁶, Kaijiong Zhang⁶, Xin Ma³, Jian Huang⁵, Dongsheng Wang⁶, Keyan Zhao³, Ping Leng¹, Huaichao Luo⁶

Affiliations

¹ Chongqing Key Laboratory of Sichuan-Chongqing Co-construction for Diagnosis and Treatment of Infectious Diseases Integrated Traditional Chinese and Western Medicine, College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
² Department of Clinical Laboratory, Guangyuan Central Hospital, Guangyuan, China.
³ GenomCan Inc., Chengdu, China.
⁴ Sichuan Cancer Hospital and Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China.
⁵ Center for Informational Biology, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
⁶ Department of Clinical Laboratory, Sichuan Cancer Hospital and Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China.

Abstract

Many studies in recent years have demonstrated that some messenger RNA (mRNA) in platelets can be used as biomarkers for the diagnosis of pan-cancer. The quantitative real-time polymerase chain reaction (RT-qPCR) molecular technique is most commonly used to determine mRNA expression changes in platelets. Accurate and reliable relative RT-qPCR is highly dependent on reliable reference genes. However, there is no study to validate the reference gene in platelets for pan-cancer. Given that the expression of some commonly used reference genes is altered in certain conditions, selecting and verifying the most suitable reference gene for pan-cancer in platelets is necessary to diagnose early stage cancer. This study performed bioinformatics and functional analysis from the RNA-seq of platelets data set (GSE68086). We generated 95 candidate reference genes after the primary bioinformatics step. Seven reference genes (YWHAZ, GNAS, GAPDH, OAZ1, PTMA, B2M, and ACTB) were screened out among the 95 candidate reference genes from the data set of the platelets' transcriptome of pan-cancer and 73 commonly known reference genes. These candidate reference genes were verified by another platelets expression data set (GSE89843). Then, we used RT-qPCR to confirm the expression levels of these seven genes in pan-cancer patients and healthy individuals. These RT-qPCR results were analyzed using the internal stability analysis software programs (the comparative Delta CT method, geNorm, NormFinder, and BestKeeper) to rank the candidate genes in the order of decreasing stability. By contrast, the GAPDH gene was stably and constitutively expressed at high levels in all the tested samples. Therefore, GAPDH was recommended as the most suitable reference gene for platelet transcript analysis. In conclusion, our result may play an essential part in establishing a molecular diagnostic platform based on the platelets to diagnose pan-cancer.

Keywords: normalization; pan-cancer; platelets; quantitative real time polymerase chain reaction; reference genes.