RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers

Abstract

Identification of appropriate reference genes (RGs) is critical to accurate data interpretation in quantitative real-time PCR (qPCR) experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq) to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.

Alternative Splicing Events as Indicators for the Prognosis of Uveal Melanoma

“…These findings also point out the way for future clinical application. As we known, ribosome biogenesis is a tightly regulated process that is critical for fundamental cellular functions including cell growth and division [26]. These ribosomal protein genes which encode structural proteins associated with ribosome biosynthesis are the most suitable for treatment in UM.…”

Clonal replacement of tumor-specific T cells following PD-1 blockade

“…Final pooled libraries were sequenced on an Illumina HiSeq 2500 with paired-end 50-bp read lengths. Paired end RNA-seq libraries from basal cell carcinoma tumors (Atwood et al ., 2015 14 ), cutaneous squamous cell carcinoma tumors (Hoang, et al , 2017 15 ) and T cell subsets (Simoni, et al . 2018 21 and this study) were aligned to the GRCh38 reference genome using STAR (version 2.6.1a) following adapter trimming by cutadapt (version 1.17).…”

“…We identified 577 genes differentially expressed between patient tumors, which included Ras signaling genes, suggesting aberrant squamous cell pathway activation in BCC, as previously reported 13 . Scoring of malignant cells for enrichment of BCC and SCC expression signatures from bulk RNA-seq 14 , 15 revealed a differentiation continuum with basal signature enrichment in nodular BCCs and squamous signature enrichment in infiltrative and metatypical BCCs ( Fig. 1i ).…”

Human Skin Equivalents Demonstrate Need for Neuro‐Immuno‐Cutaneous System

“…The silk scaffold with lipoaspirate was used as a patient‐specific model . The main advantage of using lipoaspirate, as opposed to a single‐cell source such as hASCs, is that numerous primary, human cell types are contributed from the lipoaspirate (adipocytes, preadipocytes, endothelial cells, and smooth muscle cells, immune cells including macrophages) which results in a more physiologically relevant system than a stem‐cell‐only model . However, the disadvantages include patient variability and variability in cell‐type composition which may differ from patient‐to‐patient, or even in different samples from the same patient source …”

“…Through RNASeq, numerous genes were up‐ or downregulated in a distinct manner with respect to the specific HSE study group (i.e., each HSE group had distinct gene expression) that would not have been possible to identify as quickly or thoroughly by other techniques such as real‐time reverse transcriptase polymerase chain reaction (qRT‐PCR) or microarrays due to lower sensitivity and throughput . Further, RNASeq allowed the identification of genes which may be important for development of an in vitro HSE model, and which were different with the study groups that contained neural or immune components.…”