Abstract
Dysregulation of BCL2 is a pathophysiology observed in haematological malignancies. For implementation of available treatment-options it is preferred to know the relative quantification of BCL2 mRNA with appropriate reference genes. For the choice of reference genes-(i) Reference Genes were selected by assessing variation of >60,000 genes from 4 RNA-seq datasets of haematological malignancies followed by filtering based on their GO biological process annotations and proximity of their chromosomal locations to known disease translocations. Selected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using geNorm, NormFinder, BestKeeper and RefFinder. (ii) 43 commonly used Reference Genes were obtained from literature through extensive systematic review. Levels of BCL2 mRNA was assessed by qPCR normalized either by novel reference genes from this study or GAPDH, the most cited reference gene in literature and compared. The analysis showed PTCD2, PPP1R3B and FBXW9 to be the most unregulated genes across lymph-nodes, bone marrow and PBMC samples unlike the Reference Genes used in literature. BCL2 mRNA level shows a consistent higher expression in haematological malignancy patients when normalized by these novel Reference Genes as opposed to GAPDH, the most cited Reference Gene. These reference genes should also be applicable in qPCR platforms using Taqman probes and other model systems including cell lines and rodent models. Absence of sample from healthy-normal individual in diagnostic cases call for careful selection of Reference Genes for relative quantification of a biomarker by qPCR.BCL2 can be used as molecular diagnostics only if normalized with a set of reference genes with stable yet low levels of expression across different types of haematological malignancies.
Highlights
Inspired by genome wide search for RGs from publicly available RNA-seq or microarray data in human and other organisms [23,24,25], we report here a set of novel candidate RGs obtained from an unbiased search of >60,000 genes in haematological malignancies to be used to normalize BCL2 and other anti-apoptotic genes in qPCR as molecular diagnostics
Quantification by qPCR could be the choice of pathology laboratories for a quick and cost-effective platform for single-gene expression level, with appropriate RG
In the systematic review we found a number of research articles [55,56,57,58,59,60,61,62] that have used TaqMan probes instead of SYBR green, whereas our validation experiment was carried out using SYBR green probes
Summary
Overexpression of BCL2 (B-cell lymphoma 2), a mitochondrial membrane protein has been observed in several haematological malignancies due to genetic and epigenetic mechanisms. ABL proto-oncogene 1 (ABL1), the recommended RG for gene expression studies with leukemic patients [16] was found to have extremely low expression in neutrophils [17] making it unsuitable as RG for the specific case Such discrepancies have prompted researchers to analyze gene expression across multiple tissues [18] or pan-cancer database like TCGA [19] to propose normalization factors using multiple RG candidates. Inspired by genome wide search for RGs from publicly available RNA-seq or microarray data in human and other organisms [23,24,25], we report here a set of novel candidate RGs obtained from an unbiased search of >60,000 genes in haematological malignancies to be used to normalize BCL2 and other anti-apoptotic genes in qPCR as molecular diagnostics
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