Saliva as a sampling source for the detection of leukemic fusion transcripts

Abstract

Background: Saliva has long been used as a sampling source for clinical diagnosis of oral disease such as oral squamous cell carcinoma, or therapeutic drug monitoring. The aims of this study was to ascertain if saliva RNA could be stored at room temperature and to study if saliva could be a convenient source for fusion transcripts in leukemic patients.

Methods: This is a cross-sectional diagnostic study. We first developed a Saliva RNA tube for stable storage of whole saliva RNA at room temperature. Then we detected the leukemic fusions in the whole saliva from seven leukemic patients and twenty healthy volunteers, and compared with the results obtained from the bone marrow of the patients.

Results: Human gene transcripts could be reproducibly detected in the whole saliva for at least four weeks when stored in the developed composition at room temperature. Concordant results of the fusion transcripts were obtained between the saliva and the bone marrow in the seven leukemic patients and no fusions were detected in the healthy controls.

Conclusions: The results support our hypothesis that human whole saliva could be a reliable and convenient sampling source for the detection of leukemic fusions.

Figure 1

The stability of RNA extracted from the whole saliva stored at room temperature. Saliva was collected respectively from two healthy individuals and stored in the Saliva RNA tubes for up to four weeks. Total RNA was extracted at different time intervals. The transcripts of β-actin were detected using the qRT-PCR assay at different weeks. (A) The Cq values of the the two samples at different weeks. The Cq for the male and female saliva were calculated to be 26.00 ± 0.70 and 25.34 ± 0.43, respectively. (B) The ΔCq values of the two samples at different weeks. ΔCq = Cq_RT(−) – Cq_RT(+), where Cq_RT(+) was the Cq values obtained from the extracted RNA with reverse transcription, whereas Cq_RT(−) was the Cq values obtained without reverse transcription.

Figure 2

Detection of fusion transcripts in saliva from both healthy individuals and leukemic patients. (A) qRT-PCR results of the representative healthy individuals for the detection of BCR-ABL, PML-RARα, and AML-ETO. (B) qRT-PCR results of the three types of leukemia patients for the detection of BCR-ABL, PML-RARα, and AML-ETO. The upper panel showed the pathological results for the patients and the lower panel showed the corresponding qRT-PCR results. In all detections, GUS was used as the reference gene and water was used as the no-template control (NTC).

Figure 3

Determination of the type of fusion transcripts in saliva from leukemic patients. qRT-PCR results of the CML and APL patients for the typing of BCR-ABL and PML-RARα, respectively. In all detections, GUS was used as the reference gene and water was used as the no-template control (NTC).