Reference Genes For Data Normalization Research Articles

Isoflurane preconditioning induced genomic changes in mouse cortex

BackgroundAltered patterns of genetic expression induced by isoflurane preconditioning in mouse brain have not yet been investigated. The aim of our pilot study is to examine the temporal sequence of changes in the transcriptome of mouse brain cortex produced by isoflurane preconditioning. MethodsTwelve-wk-old wild-type (C57BL/6J) male mice were randomly assigned for the experiments. Mice were exposed to isoflurane 2% in air for 1 h and brains were harvested at the following time points—immediately (0 h), and at 6, 12, 24, 36, 48, and 72 h after isoflurane exposure. A separate cohort of mice were exposed to three doses of isoflurane on days 1, 2, and 3 and brains were harvested after the third exposure. The NanoString mouse neuropathology panel was used to analyse isoflurane-induced gene expression in the cortex. The neuropathology panel included 760 genes covering pathways involved in neurodegeneration and other nervous system diseases, and 10 internal reference genes for data normalisation. ResultsGenes involving several pathways were upregulated and downregulated by isoflurane preconditioning. Interestingly, a biphasic response was noted, meaning, an early expression of genes (until 6 h), followed by a transient pause (until 24 h), and a second wave of genomic response beginning at 36 h of isoflurane exposure was noted. ConclusionsIsoflurane preconditioning induces significant alterations in the genes involved in neurodegeneration and other nervous system disorders in a temporal sequence. These data could aid in the identification of molecular mechanisms behind isoflurane preconditioning-induced neuroprotection in various central nervous system diseases.

Identification and validation of reference genes for qRT-PCR analyses under different experimental conditions in Allium wallichii

Himalayan onion (Allium wallichii) is a perennial bulbous herb with high ornamental value and has long been used as traditional medicines in Nepal and China because of the anti-cancer and anti-microbial activities. Wild Allium wallichii features different flower colors, including purple, pink, deep purple and white. However, little is known about the molecular mechanisms of color formation during A. wallichii flower development stages due to the lack of optimal reference genes. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool for quantifying expression levels of target genes. The accuracy of qRT-PCR analyses is largely dependent on the identification of stable reference genes for data normalization. The stability of reference gene expression may vary with plant species and environmental conditions. The aim of this study was to select stable reference genes for qRT-PCR analyses of target genes at flower development stages, in different flower colors and organs for Allium wallichii. The CDSs of eight potential reference genes (TUB2, ACT1, GAPC, EF1α, UBQ, UBC, SAND and CYP1) were cloned and their stability was evaluated by four programs (Delta Ct, geNorm, NormFinder and BestKeeper), and the results were further integrated into a comprehensive rank by RefFinder. The results showed that TUB2 and GAPC were the most stable two reference genes at different developmental stages of purple- and white-flower genotypes and across all samples. UBC and TUB2 expression was stable at different developmental stages of purple flowers. CYP1 and TUB2 were stably expressed at different developmental stages of white flowers. GAPC and SAND showed the highest rankings in different flower colors. TUB2 and EF1α performed the best in different tissues. ACT1 was the least stable gene in all tested samples. Moreover, DIHYDROFLAVONOL-4-REDUCTASE (DFR) gene that involved in anthocyanin synthesis was used to evaluate the effectiveness of the selected candidates. This study identified the first set of suitable reference genes for qRT-PCR analyses, which will lay the foundation for gene function study in A. wallichii.

P-517 Validating reference gene expression stability in human ovarian follicles, oocytes, cumulus cells, ovarian medulla, and ovarian cortex tissue

Abstract Study question How does the expression stability of commonly used reference genes (RG) vary in different human ovarian cells and tissues? Summary answer The RG stability differed among human ovarian cell types and tissues, but an RG with high stability was identified for each cell and tissue type. What is known already The expression of RGs used to normalize RT-qPCR may vary between different tissues, cell types, and experimental conditions. Hence, selecting the most appropriate RGs is critical in any experimental design to interpret data generated by RT-qPCR with the best accuracy. Human ovarian cells are phenotypically very different and often only available in limited amounts. In several animal species RG expression stability has been validated in oocytes and other ovarian cells, however, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. Study design, size, duration This is an experimental study performed at a university hospital from January 2021 to September 2021. Participants/materials, setting, methods The freely available NormFinder software was used to analyze the expression stability of five commonly used RGs (GAPDH, B2M, RPLP0, ACTB, and PPIA) in human oocytes (n = 160), preantral follicles (n = 160), cumulus cells (n = 13), ovarian medulla (n = 8), and ovarian cortex tissue (n = 60). Samples were collected from 29 patients (aged 28 years on average; range 14–36) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Main results and the role of chance The Normfinder software identified ACTB as the best RG for oocytes and cumulus cells; and B2M for medulla tissue and isolated follicles. Overall, comparisons of the cycle threshold (Ct) values demonstrated a wide variation among the RGs within the same group of samples (P < 0.05), especially for oocytes and preantral follicles with normalized RNA. The genes ACTB and RPLP0 showed the highest levels of expression and PPIA the lowest levels of expression in all types of samples, except for cortex tissue, where PPIA had the highest level of expression and B2M the lowest. These results infer that different results could be obtained when using different RGs for data normalization. The combination of two RGs only marginally increased stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For cultured ovarian cortex culture, GAPDH or ACTB were found to be the most stable genes depending on culture conditions. Limitations, reasons for caution Since human oocytes are scarce and contain a small amount of RNA, only five RGs were evaluated. Moreover, only germinal vesicle (GV) oocytes and preantral follicles (60-80 µm) were tested. Future studies may validate other genes and investigate the effect of oocyte maturation and follicle growth on RG stability. Wider implications of the findings Our findings highlight the importance of validating RGs for each cell type or tissue and culture condition. Hence, our results can be of use as guidance for future studies involving gene expression analyses in human ovarian cells and tissues, including oocytes and preantral follicles. Trial registration number Not applicable

Identification and Validation of Reference Genes for RT-qPCR Normalization in Nauphoeta cinerea (Olivier, 1789) (Blattodea, Blaberidae)

Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows large-scale analysis of very small changes in gene expression. For the calculation of gene expression, such as the delta-delta Ct method, different PCR primer efficiencies (E) may affect the result, as PCR primer yields are assumed to be comparable for the gene of interest and housekeeping gene. Therefore, identification of a suitable reference gene for data normalization is an important step in the development of qPCR assays. Furthermore, accurate and reliable results depend on the use of stable reference genes for normalization. The aim of the current study is the identification and validation of a set of six housekeeping genes (GADPH, RPS18, α-TUB, EF1α, ArgK, and ACTB) in cockroach species Nauphoeta cinerea adults using five different algorithms (ΔCt method, Bestkeeper, geNorm, Normfinder and RefFinder) to evaluate the stability of selected reference genes expression. Our results show that α-Tub use provides accurate normalization of gene expression levels in N. cinerea adults. In addition, since the GADPH is selected as the second most stable reference gene, GADPH can be also used for transcript analysis N. cinerea adults. Our study also showed that ACTB (β-actin) should not be used for normalizing transcript levels when examining N. cinerea adults. Additionally, validation studies for reference genes in cockroaches are very few (only one) in the literature. Therefore, the results highlight the need for validation of reference genes under biotic and abiotic conditions in q-RT-PCR studies in cockroaches.

Evaluation of optimal reference genes for the normalization by qPCR in viable but nonculturable state in Xanthomonas campestris pv. campestris

AbstractXanthomonas campestris pv. campestris (Xcc), the pathogen of black rot of crucifers, could be induced into the viable but nonculturable (VBNC) state under stress, such as being induced by low concentration of Cu2+ and acid condition induction. The main objective of current study was to select the optimal reference genes for data normalization by qPCR in the VBNC state of Xcc. In this study, Xcc was induced into VBNC state by Cu2+ with an initial concentration of OD600 = 0.18 and OD600 = 0.45, and all viable bacterial cells lost the culturability and entered into the VBNC state at 24 h and 48 h respectively. The concentration of VBNC cells was 3.3 × 107 cells/ml and 1.5 × 107 cells/ml respectively. Eight candidate reference genes (gyrB, pbpA, gapA, rpoB, tufA, 16S rRNA, ugpC and recA) were selected to assess the expression stability in the VBNC state of Xcc by four algorithms (geNorm, NormFinder, delta‐Ct and BestKeeper), while RefFinder was used to integrate the output from the four algorithms in fine. According to the algorithm analysis and validation of clpX expression analysis, the most suitable reference gene this study identified was the combination of ugpC and pbpA, and the least suitable gene was 16S rRNA in the Xcc VBNC state. In this study, we identified the most stable internal controls under Cu2+‐inducing conditions for calibration qRT‐PCR analyses of Xanthomonas campestris pv. campestris and will be helpful to explore the stress resistance mechanism in this bacterium.

Evaluation of reference genes for RT-qPCR gene expression analysis in Arabidopsis thaliana exposed to elevated temperatures.

Increases in environmental temperature are directly linked to the issue of climate change and are known to significantly disrupt plant growth and development. Studies of gene expression in plants commonly include RT-qPCR but the reliability of the method depends on the use of suitable reference genes for data normalization. Despite this, no reference genes have been validated specifically for experiments in Arabidopsis thaliana employing treatments with elevated temperature. Here, ten genes were selected for expression stability analysis based on the screening of available literature and microarray data from temperature-treated A. thaliana. Expression levels of candidate reference genes were measured in 12-day-old seedlings, rosette leaves and flower buds of 5-week-old A. thaliana plants exposed to five different temperatures (22°C, 27°C, 32°C, 37°C and 42°C) and their expression stabilities were assessed using four statistical algorithms (BestKeeper, geNorm, NormFinder and comparative ΔCq method). This study provides reliable reference genes for use in A. thaliana RT-qPCR expression analyses employing elevated temperature treatments, namely OGIO and PUX7 in seedlings, UBC21 and PUX7 in leaves, TIP41 and UBC21 in buds, and TIP41 and UBC21 in all three tissues combined. Orthologues of these genes can be of potential use in less studied plants, especially agricultural species heavily affected by climate change.

Stable Reference Gene Selection for qRT-PCR Normalization in Strawberry (Fragaria × ananassa) Leaves under Different Stress and Light-Quality Conditions

Selecting an appropriate reference gene is of crucial importance for improving the accuracy of qRT-PCR analyses. In this study, strawberry (Fragaria ananassa) seedlings were subjected to different environmental conditions including heat, cold, drought, salt, white-light, blue-light, and red-light treatments. The expression levels of seven candidate reference genes, including Fa18S, FaGAPDH, FaPIRUV, FaDBP, FaHISTH4, FaACTIN1, and FaACTIN2, in the strawberry leaves were measured by qRT-PCR. Then, four programs (geNorm, NormFinder, BestKeeper, and RefFinder) were employed as tools to evaluate the expression stability of the candidate reference genes. The results showed that the expression stability of the reference genes varied under different conditions. For the cold stress and white-light treatments, FaACTIN2 was evaluated to be the most stable reference gene. FaGAPDH should be used as the reference gene under salt-stress condition and red-light treatment. For the data normalization under drought-stress treatment, FaDBP is the recommended reference gene with the highest expression stability. FaHISTH4 was observed to be the best reference gene for data normalization under heat stress and blue-light treatment. This work provides information on selecting reference genes for accurate gene expression analyses of target genes in strawberry leaves under various abiotic stress and light-quality conditions.

Identification and validation of reference genes for RT-qPCR analysis in fetal rat pancreas

The choice of reference gene is crucial for quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay. To screen and determine the suitable reference genes in fetal rat pancreas, we selected eight candidate reference genes (Gapdh, Actb, Rn18 s, B2m, Rpl13a, Tbp, Ywhaz and Ubc), and evaluated the constancy of gene expression from fetal rat pancreases in non-pathological situation and prenatal dexamethasone exposure (PDE) model, using four algorithms: GeNorm, NormFinder, Bestkeeper and Comparative ΔCt method. In addition, the alteration of mRNA levels of pancreatic insulin was compared between control and PDE groups to validate the reliability of selected reference genes for data normalization of RT-qPCR. The comprehensive ranking of reference genes under physiological condition was as follow: Gapdh >Actb >Ywhaz >Ubc >Rn18s >Rpl13a >B2m >Tbp (female); Actb >Ywhaz >Gapdh >Ubc >B2m >Rpl13a >Rn18 s | Tbp (male). The top ranking reference genes were also stably expressed in PDE fetal pancreas. The best reference gene combinations are: Ywhaz+Actb for female and Ywhaz+Gapdh for male fetal rat pancreas, respectively. Compared with low ranking or single reference gene, the change trend of insulin mRNA normalized by the best reference gene combination between control and PDE groups was more significant and consistent with that of serum insulin level. In conclusion, our results provided the optimal combination of stable reference genes for RT-qPCR assay in pancreatic developmental toxicity study.

Validation of common reference genes stability in exosomal mRNA-isolated from liver and breast cancer cell lines.

Accurate relative gene expression analysis by reverse transcription-quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well-known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes-isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK-BR-3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes-isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

LRIG1 expression and colorectal cancer prognosis

BackgroundTo make the right treatment decisions about colorectal cancer (CRC) patients reliable predictive and prognostic data are needed. However, in many cases this data is not enough. Some studies suggest that LRIG1 gene (leucine-rich repeats and immunoglobulin-like domains1) has prognostic implications in different kinds of cancers.MethodsOne hundred and two patients with colorectal cancer were retrospectively analyzed for LRIG1 expression at both mRNA and protein levels. SYBR Green Real-Time RT-PCR technique was used for mRNA expression analyses and Glyceraldehyde-3-Phosphate Dehydrogenase gene (GAPDH) was considered as a reference gene for data normalization. LRIG1 protein expression was analyzed using Immunohistochemistry. Additionally, appropriate statistic analyses were used to assess the expression of LRIG1 in test and control groups. The prognostic significance of LRIG1 expression was analyzed using the univariate and multivariate analyses.ResultsThe data revealed that the expression of LRIG1 in both mRNA and protein levels was down regulated in colorectal tumor tissues (P < 0.01) but is not clinically relevant prognostic indicator in CRC.ConclusionsTherefore, it is suggested that LRIG1 expression analyses may not be considered as an important issue when making informed and individualized clinical decisions regarding the management of colorectal cancer patients.

Reference gene selections for real time quantitative PCR analysis of gene expression in different oat tissues and under salt stress

Appropriate choice of reference genes for data normalization is of critical importance for accurate real time reverse transcription quantitative PCR (RT-qPCR) analysis of gene expression. Oat is an agriculturally important crop cultivated widely around the world for grain or forage, and appropriate reference genes for reliable gene expression analysis remain to be identified. In this study, we selected nine candidate reference genes based on available oat RNA-seq data. We then conducted a systematic evaluation of the relative expression stability of these genes in different tissues and under salt stress using statistical algorithms, geNorm, NormFinder, and BestKeeper. Our findings reveal that the highest-ranked reference genes for accurate data normalization should be selected according to specific sample subsets. For different tissues, the combination of two reference genes [elongation factor 1-alpha (EF1α) and serine/threonine protein phosphatase 2] was sufficient for accurate normalization. For salt stress treatment, the combination of two reference genes (EF1α and TATA-binding protein) was sufficient for accurate normalization. Moreover, the commonly used reference genes, actin and glyceraldehyde 3-phosphate dehydrogenase, were least suitable for data normalization of oat samples. Expression of a salt stress-inducible transcription factor Avena sativa WRKY2 was investigated to validate the reference genes identified in this study. This is the first systematic study of reference gene selection in cultivated oat and provides guidelines to obtain more accurate RT-qPCR results in this species.

Abstract 5963: NGS-based murine pan-cancer gene expression panel for immuno-oncology and tumor microenvironment studies

Abstract INTRODUCTION: Cancer is a heterogeneous disease and highly related to the immunological system. The significance of fully understanding the interactions between cancer and immunity in cancer therapeutics has been broadly noted, particularly for immunotherapies. Experimental murine models derived from a variety of biological technologies are essential preclinical model systems utilized before clinical studies. Molecular pathology techniques, such as gene expression biomarkers, are widely used to systematically profile and characterize these models. So far, however, there has been no robust and cost-effective assay designed for murine clinical immuno-oncology (I/O) research to characterize tumor-immune interactions at a gene expression level. METHODS: Marker genes were identified by combining public and in-house reference genomic data for non-tumor bearing mice as well as tumor mouse models. Oligonucleotide-based hybridization/capture techniques analogous to exome sequencing, targeting specific regions are well established on Illumina Novaseq and BGI MGIseq platforms. These methods were applied to the murine I/O panel assay, with accuracy and reproducibility verified across these two sequencing platforms. The results were further qualified using a PCR array platform. RESULTS: A mouse gene panel involving 1080 genes that encompass surface markers and transcriptomic biomarkers for the immune system, key pathways at the interface of the tumor, tumor microenvironment (TME), and immune response, as well as internal reference genes for data normalization has been established. By combining advanced NGS technologies and specific bioinformatic pipelines, we were able to detect and distinguish various immune and stromal cells involved in the complex interaction between the tumor and its microenvironment, allowing for a multifaceted characterization of disease biology and the interrogation of immune response and evasion. We validated the sensitivity, specificity, and repeatability of the panel by qPCR experiments. CONCLUSIONS: We have established a murine-I/O NGS panel involving 1080 genes to characterize tumor-immune interactions more efficiently and in a cost-effective manner for preclinical studies. This provides an enhanced insight into the TME, with high-content data that has the potential to translate more effectively to the clinic as well as for early identification of immunotherapy companion diagnostics. Citation Format: Xiaobo Chen, Jia Xue, Qixiang Li, Sheng Guo. NGS-based murine pan-cancer gene expression panel for immuno-oncology and tumor microenvironment studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5963.

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the main insect pest of peppers (Capsicum spp.) throughout the southern U.S. and a potential target for novel control methods that may require gene expression analyses. Careful selection of adequate reference genes to normalize RT-qPCR data is an important prerequisite for gene expression studies since the expression stability of reference genes can be affected by the experimental conditions leading to biased or erroneous results. The lack of studies on validation of reference genes for RT-qPCR analysis in A. eugenii limits the investigation of gene expression, therefore it is needed a systematic selection of suitable reference genes for data normalization. In the present study, three programs (BestKeeper, geNorm and NormFinder) were used to analyze the expression stability of candidate reference genes (β-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) in A. eugenii under different experimental conditions. Our results revealed that the most stably expressed reference genes in A. eugenii varied according to the experimental condition evaluated: developmental stages (EF1-α, 18S and RPL12), sex (RPS23 and RPL12), low temperature (GAPDH and α-TUB), high temperature (α-TUB and RPS23), all temperatures (α-TUB and GAPDH), starvation (RPL12 and α-TUB), and dsRNA exposure (α-TUB and RPL12). Our study provides for the first time valuable information on appropriate reference genes that can be used in the analysis of gene expression by RT-qPCR in biological experiments involving A. eugenii.

Reference gene selection and myosin heavy chain (MyHC) isoform expression in muscle tissues of domestic yak (Bos grunniens)

Domestic yak (Bos grunniens) is the most crucial livestock in the Qinghai-Tibetan Plateau, providing meat and other necessities for local people. The skeletal muscle of adult livestock is composed of muscle fibers, and fiber composition in muscle has influence on meat qualities, such as tenderness, pH, and color. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful tool to evaluate the gene expression of muscle fiber, but the normalization of the data depends on the stability of expressed reference genes. Unfortunately, there is no consensus for an ideal reference gene for data normalization in muscle tissues of yak. In this study, we aimed to assess the stability of 14 commonly used candidate reference genes by using five algorithms (GeNorm, NormFinder, BestKeeper, Delat Ct and Refinder). Our results suggested UXT and PRL13A were the most stable reference genes, while the most commonly used reference gene, GAPDH, was most variably expressed across different muscle tissues. We also found that the extensor digitorum lateralis (EDL), trapezius pars thoracica (TPT), and psoas major (PM) muscle had the higher content of type I muscle fibers and the lowest content of type IIB muscle fibers, while gluteobiceps (GB) muscle had the highest content of type IIB muscle fibers. Our study provides the suitable reference genes for accurate analysis of yak muscle fiber composition.

Identification and evaluation of reference genes for gene expression analysis in the weevil pest Pagiophloeus tsushimanus using RT-qPCR

Abstract Pagiophloeus tsushimanus is a newly and specialist wood-boring beetle of Cinnamomum camphora in China. RT-qPCR is an accurate quantitative method to quantify target genes expression, which relies on suitable reference genes for data normalization. Reference genes must to be stably expressed under specific experimental conditions. No suitable reference genes of P. tsushimanus have been reported so far. Therefore, it is necessary to identify and evaluate suitable reference genes for the study of functional genes of this pest. In this research, the expression stability of eight candidate reference genes (RPS3, 18S rRNA, GAPDH, TBP, RPL10, UBQ, GST, and RPS27A) were systematically evaluated in P. tsushimanus by five algorithms (geNorm, BestKeeper, NormFinder, delta Cq, and RefFinder) under different developmental stages, various tissues, and insects reared on different plants, and validated by the olfactory key gene odorant binding protein 33 (PtsuOBP33). The results showed that three stable reference genes combination were necessary for quantitative analysis of target gene. RPS3, RPL10, and UBQ were the optimal reference genes combination for gene expression analysis of developmental stages, while RPL10, RPS3, and 18S rRNA were recommended for different tissues, and 18S rRNA, TBP, and RPS3 were recommended for insects reared on different plants. The results indicated that suitable reference genes should be screened out for gene expression analysis under different conditions. This paper systematically analyzed and obtained suitable reference genes in P. tsushimanus for the first time, which would contribute to the functional analysis of genes and the in-depth mining of genetic resources in it.

Gene Expression Analysis in Bacteria by RT-qPCR.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.

Validation of Reliable Reference Genes for RT-qPCR Studies of Target Gene Expression in Colletotrichum camelliae During Spore Germination and Mycelial Growth and Interaction With Host Plants.

The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. Colletotrichum camelliae Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of C. camelliae, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in C. camelliae thus far. In this study, 12 candidate genes (i.e., CcSPAC6B12.04c, CcWDR83, Cchp11, Ccnew1, CcHplo, CcRNF5, CcHpcob, CcfaeB-2, CcYER010C, CcRNM1, CcUP18, and CcACT) were isolated from C. camelliae and assessed as potential reference genes. The expression stability of these genes in C. camelliae during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that Cenew1, CcHplo, and CcSPAC6B12.04c were the most stable reference genes in C. camelliae under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of C. camelliae.

Selection of reliable reference genes for gene expression analysis in seeds at different developmental stages and across various tissues in Paeonia ostii.

Paeonia ostii seeds have recently been identified as a new source of α-linolenic acid in China. Studying the gene expression patterns of unsaturated fatty acid-related genes would be helpful for understanding the mechanism of α-linolenic acid accumulation. Quantitative real-time polymerase chain reaction (qRT-PCR) is a useful method for reliably evaluating gene expression, and it is necessary to select reliable reference genes for data normalization in qRT-PCR analysis. In this study, we evaluated the expression stability of 12 candidate reference genes using four mathematical algorithms (∆Ct, BestKeeper, NormFinder, and geNorm). The web-based tool RefFinder was used to integrate the results and to provide a comprehensive ranking order. The expression stability ranking orders of reference genes were different caculated by these four algorithms, and the ranking order analyzed by the RefFinder was UBQ > Tip41 > UCE > EF-1α > α-TUB > PP2A > ACT > GAPDH > SAM > CYP > β-TUB > 18S at the different seed development stages, and UBQ > Tip41 > EF-1α > α-TUB > PP2A > UCE > GAPDH > SAM > ACT > CYP > 18S > β-TUB in P. ostii tissues. UBQ and Tip41 are the two most stable whereas 18S and β-TUB are the two least stable reference genes for gene expression in various tissues and seeds at different developmental stages in P. ostii.

Simplified method for applying static isotropic tensile strain in cell culture experiments with identification of valid RT-qPCR reference genes for PDL fibroblasts.

Periodontal ligament fibroblasts (PDLF) play an important mediating role in orthodontic tooth movement expressing various cytokines, when exposed to compressive or tensile strain. Here, we present a simplified and easy-to-handle, but reliable and valid method for simulating static isotropic tensile strain in vitro using spherical silicone cap stamps. Furthermore, we identify appropriate reference genes for data normalization in real-time quantitative polymerase chain reaction (RT-qPCR) experiments on PDLF subjected to tensile strain. PDLF were cultivated on flexible bioflex membranes and exposed to static isotropic tensile strain of different magnitudes and timeframes. We determined cell number, cytotoxicity, and relative expression of proinflammatory genes cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). For normalization of RT-qPCR data, we tested the stability and validity of nine candidate reference genes with four mathematical algorithms (geNorm, NormFinder, comparative ΔCq, and BestKeeper) and ranked them based on their calculated expression stability. We observed no decrease in cell number or cytotoxic effect at any of the applied magnitudes and timeframes of tensile strain. At 16 per cent and 35 per cent tensile strain for 48 hours, we detected a significant increase in COX-2 and decrease in IL-6 gene expression. Highest stability was found for TBP (TATA-box-binding protein) and PPIB (peptidylprolyl isomerase A) in reference gene validation. According to the geNorm algorithm, both genes in conjunction are sufficient for normalization. In contrast to all other candidate genes tested, gene expression normalization of target gene COX-2 to reference genes EEF1A1, RPL22, and RNA18S5 indicated no significant upregulation of COX-2 expression. A strain magnitude of 16 per cent for 48 hours elicited the most distinct cellular response by PDLF subjected to static tensile isotropic strain by the presented method. TBP and PPIB in conjunction proved to be the most appropriate reference genes to normalize target gene expression in RT-qPCR studies on PDLF subjected to tensile strain.

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation.

Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.