Identification and validation of the optimal reference genes for standardizing the gene expression profiling diagnostic panel of Ph-like B-lineage acute lymphoblastic leukemia

AbstractGene expression profiling is the criterion standard for recognizing Ph-like ALL signatures among B-ALLs. The prerequisite of GEP is the accurate normalization of target genes with stable expression of housekeeping genes in a quantitative PCR. HKGs exhibit differential expression in the different experimental conditions and affect the target genes' expression, leading to imprecise qPCR results. The selection of stable HKGs is crucial in GEP experiments, especially in identifying high-risk Ph-like ALL cases. We have evaluated the expression stability of nine HKGs (GAPDH, ACTB, GUSB, RNA18S, EEF2, PGK1, B2M, TBP andABL1) in identified Ph-like ALLs and Ph-negative (n = 23 each) using six algorithms, 4 traditional softwares; geNorm, BestKeeper, NormFinder, Delta Cq value method, and two algorithms, RefFinderTM and ComprFinder. Further, we have validated the expression of 8 overexpressed normalized genes in Ph-like ALL cases (JCHAIN, CA6, MUC4, SPATS2L, BMPR1B, CRLF2, ADGRF1 and NRXN3). GeNorm, BestKeeper, NormFinder, Delta Cq value method, RefFinderTM and ComprFinder algorithm analysis revealed thatEEF2, GAPDH, andPGK1 form the best representative HKGs in Ph-like ALL cases, while RNA18s, ß-actin, andABL1 in Ph-negative ALLs. Lastly, we performed a correlation analysis and found that the combination ofEEF2, GAPDH, andPGK1 represents the best combination with a very high correlation in Ph-like ALL cases. This is the first report that showsEEF2, GAPDH, andPGK1 are the be...
Source: Clinical and Experimental Medicine - Category: Research Source Type: research