Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

Background: Monocytes and macrophages are essential components of the innate and adaptive immune responses and play a critical role in sepsis. Sepsis is a life-threatening organ dysfunction associated with an unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. There is a lack of studies regarding human monocytes and sepsis. This study aimed to determine the most stable internal gene (s) to investigate gene expression in monocytes/macrophages of septic patients.Methods: The expression stability of fifteen commonly used reference genes was analyzed by determining the comparative threshold cycle (Ct) values, using the BestKeeper, GeNorm, and NormFinder algorithms.Results: BestKeeper analysis revealed that the syntaxin 5 (STX5A) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes were highly stable. GeNorm pointed out STX5A and phosphoglycerate kinase 1 (PGK1) as the most suitable combination whereas through NormFinder glyceraldehyde 3- phosphate dehydrogenase (GAPDH) and 14-3-3 zeta/delta protein (YWHAZ) was the most stable combination. All programs analysis discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the least stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patients monocytes. In the case of choosing one single reference gene, the results point out to STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in monocytes/macrophages from septic patients.

Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes

Quantitative reverse transcription PCR (qRT-PCR) has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1) and nitric oxide synthase (NOS), play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1), actin 1 (Act) and ribosomal protein S7 (S7) genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.

Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging

The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P > 0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P = 0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.