Background

The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata.

Results

The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments.

Conclusions

We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.

The present case report describes a case of syphilitic lymphadenopathy and raises the awareness of the differential diagnosis of cervical lymphadenopathy. A 50-year-old male worker presented with a 6-month history of enlarged and growing lymph nodes in the right upper neck and a blood-tinged post-nasal drip. Physical examination showed multiple enlarged lymph nodes located in the right upper neck. On nasopharyngoscopy, a mass was found in the nasopharynx. The histopathology of both the nasopharyngeal mass and the enlarged lymph nodes revealed non-specific inflammation. Rapid plasma reagin test results (titer, 1:1280) and Treponema pallidum particle assay results (titer, 1:2560) were positive. Subsequently, a diagnosis of syphilis was confirmed clinically and serologically. The reaction after penicillin treatment further confirmed the syphilis diagnosis. Thus, syphilis should be considered as a possibility in the differential diagnosis of cervical lymphadenopathy.

The present study aimed to assess the impact of post-surgical hormone replacement therapy (HRT) on life quality and prognosis in women with ovarian malignancy. HRT (Premarin, Nilestriol and medroxyprogesterone) was administered following surgery in 31 patients with ovarian cancer. A total of 44 ovarian cancer patients of similar age, clinical stage and pathological features did not receive HRT following surgery. The expression of estrogen receptor (ER)-α, ERβ and progesterone receptor (PR) in cancer tissues was detected by immunohistochemical staining. Serum levels of calcitonin (CT) and transforming growth factor (TGF)-α were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Data were analyzed using Kaplan-Meier survival curves, a log-rank test and a Cox scale risk model. Quality of life was assessed in the patient groups and in healthy post-menopausal women (control) based on a questionnaire developed by the European Organization of Research and Treatment of Cancer (EORTC-C30), as well as our own specific questionnaire. A log-rank test revealed no difference in survival between the patients with and without HRT (p>0.05), and a Cox model showed that HRT was not an independent prognostic factor. The accumulated survival rate did not differ significantly based on the expression of ERα, ERβ or PR in patients with or without HRT (p>0.05). The serum TGFα levels prior to and following surgery were not significantly different in either of the two patient groups (p>0.05). Serum CT levels were higher in patients without HRT at 1.5 years following surgery (p<0.05), but no significant difference was found in the serum CT levels of patients receiving HRT. The HRT and non-HRT groups differed significantly with regard to the body and emotional functional sub-scales of the EORTC-C30 (p<0.05) and the sex quality and autonomic nerve maladjustment categories of our specific questionnaire (p<0.05). Findings of this study showed that HRT administered following surgery exhibited no apparent negative effect on prognosis in patients with ovarian cancer, regardless of ERα, ERβ or PR expression in cancer tissues, and had no effect on serum transforming growth factor (TGF)-α levels. Post-surgical HRT aided in the stabilization of serum CT levels and improved the quality of life in these patients.