Identification and Validation of Novel Reference Genes in Acute Lymphoblastic Leukemia for Droplet Digital PCR.

Josselen Carina Ramírez-Chiquito,Eleazar Israel Pérez-López,Vanessa Villegas-Ruíz,Sergio Juárez-Méndez,Kattia Alejandra Castro-López,Isabel Medina-Vera,Victoria Estefanía Saucedo-Tepanecatl,Karina Olmos-Valdez

doi:10.3390/genes10050376

Abstract

Droplet digital PCR is the most robust method for absolute nucleic acid quantification. However, RNA is a very versatile molecule and its abundance is tissue-dependent. RNA quantification is dependent on a reference control to estimate the abundance. Additionally, in cancer, many cellular processes are deregulated which consequently affects the gene expression profiles. In this work, we performed microarray data mining of different childhood cancers and healthy controls. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). Gene expression was validated in 40 acute lymphoblastic leukemia samples by means of droplet digital PCR. We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. However, we observed excellent correlations among the new reference genes (p < 0.0001). We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer.

Highlights

RNA is a very versatile molecule and its abundance is tissue-dependent
We observed differences in Gene expression (GE) between classical reference genes (CRGs) by sample. These results suggest that the droplet digital PCR (ddPCR) assay allowed us to discriminate genes with low and and new reference genes (NRGs) by sample
Our results showed that ACTB was the most highly expressed gene, with the following relationships: 1:2.1, 1:6.4, 1:8.3, 1:73, 1:88, 1:263, 1:404, ACTB:GAPDH, ACTB:RPL4, ACTB:RPS18, ACTB:UQCR2, ACTB:PSMB6, ACTB:UBQLN2 and

Summary

Introduction

RNA quantification is dependent on a reference control to estimate the abundance. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer. RNA expression has been used as a molecular marker in diverse malignancies, such as mendelian disorders [14], tuberculosis [15] and some types of cancer [16]. It is very important to establish methods for RNA quantification and reference genes (calibrator or housekeeping) that do not show variations between patients and healthy conditions

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Conclusion