Evaluation and validation of internal control genes for studying gene expression in the dinoflagellate Prorocentrum minimum using real-time PCR

Housekeeping genes (HKGs) are required for the normalization of expression levels in real-time PCR, and their selection is critical for gene expression studies. However, stable expressions of candidate HKGs vary among organisms and tissues and even according to environmental conditions. Here, we evaluated the gene expressions of 10 frequently used HKGs, including 18S rRNA, P2, ACT, TUA, TUB, CYC, eIF4E, MDH, UBQ, and GAPDH, with a total of 17 RNA samples extracted from the dinoflagellate Prorocentrum minimum. All the RNAs were prepared from various cells cultured under different conditions, including thermal shocks, toxic chemical exposures, and different life stages. Via C(T) analyses of the 10 HKGs using the geNorm software, TUA was selected as the most stable HKG for gene expression studies of the dinoflagellate, followed by MDH. Pair-wise variation (V) analysis showed that at least 2 genes were required for accurate normalization of gene expression studies depending on the experimental conditions. These HKGs are useful internal controls for the normalization of gene expression in P. minimum.     

Translational regulation by non-protein-coding RNAs: different targets, common themes

There are several classes of small non-protein-coding RNA (npcRNA) that play important roles in cellular metabolism including mRNA decoding, RNA processing and mRNA stability. Indeed, altered expression of some of these npcRNAs has been associated with cancer, neurodegenerative diseases such as Alzheimer’s disease, as well as various types of mental retardation and psychiatric disorders. The basis of this correlation is currently not understood. However, recent studies have begun to shed light on one of the mechanism(s) by which these RNAs exert their effects, namely, translational control. These data provide hope that rational treatments for these varied disorders may be in sight. Here, we review this new body of work.     

Two primate-specific small non-protein-coding RNAs in transgenic mice: neuronal expression, subcellular localization and binding partners

In a rare occasion a single chromosomal locus was targeted twice by independent Alu-related retroposon insertions, and in both cases supported neuronal expression of the respective inserted genes encoding small non-protein coding RNAs (npcRNAs): BC200 RNA in anthropoid primates and G22 RNA in the Lorisoidea branch of prosimians. To avoid primate experimentation, we generated transgenic mice to study neuronal expression and protein binding partners for BC200 and G22 npcRNAs. The BC200 gene, with sufficient upstream flanking sequences, is expressed in transgenic mouse brain areas comparable to those in human brain, and G22 gene, with upstream flanks, has a similar expression pattern. However, when all upstream regions of the G22 gene were removed, expression was completely abolished, despite the presence of intact internal RNA polymerase III promoter elements. Transgenic BC200 RNA is transported into neuronal dendrites as it is in human brain. G22 RNA, almost twice as large as BC200 RNA, has a similar subcellular localization. Both transgenically expressed npcRNAs formed RNP complexes with poly(A) binding protein and the heterodimer SRP9/14, as does BC200 RNA in human. These observations strongly support the possibility that the independently exapted npcRNAs have similar functions, perhaps in translational regulation of dendritic protein biosynthesis in neurons of the respective primates.     

Evaluation of reference genes for real-time quantitative PCR studies in

ABSTRACT: 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, EF1a, 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-[increment][increment]CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-[increment][increment]CT 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.     

Variable expression of some "housekeeping" genes during human keratinocyte differentiation

We investigated the expression levels of four cellular "housekeeping" genes during epithelial differentiation. Differentiation is a dynamic process and various cellular RNAs have been targeted for use as internal controls during differentiation of human keratinocytes, but the consistent expression of such standards has not been previously validated. We used the organotypic (raft) culture system to grow stratified and differentiated epithelium in vitro. We compared cellular RNAs from epithelial tissues of both normal human keratinocytes and keratinocytes whose differentiation scheme is altered by the replication of human papillomavirus. Using ribonuclease protection assays to quantify RNA expression levels, we found that beta-actin and glyceraldehyde-3-phosphate dehydrogenase levels fluctuated during epithelial differentiation, whereas cyclophilin RNA and 28S-ribosomal RNA were the most consistently expressed during epithelial differentiation. These stably expressed cellular RNAs can be targeted as controls to permit quantitative expression analyses of cellular and pathogen RNAs during epithelial differentiation under various experimental conditions.     

Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm (Diabrotica virgifera virgifera)

Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.     

Identification of Novel Reference Genes Using Multiplatform Expression Data and Their Validation for Quantitative Gene Expression Analysis

Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required.