Real-time PCR as a tool for quantitative analysis of PI-PLCβ1 gene expression in myelodysplastic syndrome

Abstract

Phosphoinositide-specific phospholipase C (PI-PLC) β1 is a key enzyme in nuclear signal transduction, and it is involved in many cellular processes, such as proliferation and differentiation. In particular, the involvement of the PI-PLCβ1 gene in erythroid differentiation lead us to investigate this gene in patients affected by high-risk myelodysplastic syndrome (MDS). By using fluorescence in situ hybridization (FISH) analysis, we have previously evidenced that, in MDS patients with normal GTG banding and a fatal outcome, the PI-PLCβ1 gene undergoes monoallelic and interstitial deletion. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. In the present study, we have performed a relative quantification real-time polymerase chain reaction (PCR) analysis on all of the MDS patients tested for FISH analysis. Furthermore, we have evaluated the expression of the PI-PLCβ1 gene on healthy donors and the HL60 cell line, which is useful for testing the accuracy of the technology because of its low expression of PI-PLCβ1. To analyze and quantify the levels of the two different splicing variants of PI-PLCβ1 gene (1a and 1b), we have used a TaqMan isoform specific probe. We have seen that all of the MDS patients have higher levels of the PIPLCβ1 mRNA compared to the HL60 cell line as expected, but lower levels compared to the healthy donors. Furthermore, MDS blasts always express higher levels of PI-PLCβ1b mRNA compared to PI-PLCβ1a mRNA. Our data support the contention that the deletion of the PI-PLCβ1 gene is indeed responsible for a reduced expression of the enzyme. In addition, the splicing isoform 1b, which is only nuclear, seems to be somehow partially preserved compared to the 1a isoform, which is nuclear and cytoplasmatic, hinting at a possible imbalance of the nuclear versus cytoplasmatic PI-PLC signaling which, in turn, could affect the cell cycle progression of MDS blasts.