Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in Sesamia inferens (Lepidoptera: Noctuidae)

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (−8, −6, −4, −2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.     

红掌佛焰苞基因qRT-PCR分析中内参基因的筛选

为筛选红掌(Anthurium andraeanumLinden)中稳定表达、可用于佛焰苞中实时荧光定量PCR分析(qRT-PCR)的内参基因,对5个组成型表达基因EF1-a、UBQ7、ACTB、GADPH、His3进行表达稳定性分析,并利用所筛选的内参基因研究红掌的二氢黄酮醇还原酶基因(dfr)的表达水平。结果表明,5种内参基因在不同品种间的表达稳定性不同。据内参基因标准化因子的配对差异分析(Vn/n+1),判定内参基因的最适数目为2,ACTB和UBQ7在红掌不同品种及佛焰苞发育不同阶段中表达均稳定,是理想的内参基因。dfr在不同品种的佛焰苞及佛焰苞发育过程中均有表达,且dfr表达水平的变化趋势一致,因此,所选内参基因是合适的。     

Validation of reference genes in the feline endometrium

The aim of the study was to find the most stable reference genes from: ACTB, GAPDH, RPL30, CYC, RPL17, RPS7 and YWHAZ in the feline endometrium. Three free software packages, geNorm, NormFinder and BestKeeper were used. In geNorm analysis, the most stable gene was RPS7 (at a primer concentration 1000 nM) or YWHAZ (500 and 250 nM). According to NormFinder and BestKeeper, ACTB (at all examined primer concentrations) followed by RPS7 and CYC were the most stable genes. Based on geNorm results at least two genes from among RPS7, RPL30, ACTB or YWHAZ should be chosen for Real Time-PCR result normalization.     

Validation of reference genes for quantitative real-time polymerase chain reaction assay of honeybee under various pesticide treatment conditions

In quantitative real-time polymerase chain reaction (qRT-PCR), target gene expression levels are normalized to internal reference gene(s) that are stably expressed across different conditions to determine whether they are up- or down-regulated. Therefore, it is essential to select appropriate reference gene(s) for the accurate comparison of target gene expression across different experimental conditions. Honeybee colonies can be damaged due to pesticide exposure, resulting in a decline of their population. Determination of gene expression levels is important for understanding the physiological response of honeybees to pesticide exposure. Therefore, in this study, we used qRT-PCR to analyze the expression stability of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAB1a) in honeybees subjected to treatment with different dosages and exposure durations of seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin) using four programs (geNorm, NormFinder, BestKeeper, and RefFinder). Subsequently, the expression levels of the target genes (PER, FOR, and EGR1) were calculated using different normalization methods and compared. Based on our collective results, we propose RPS5 as the most appropriate reference gene for the normalization of target gene expression levels in qRT-PCR assays for honeybees under various conditions of pesticide exposure, including pesticide type, exposure duration, and concentration.     

What is beyond a qRT‐PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes

In the last years, mesenchymal stem cells (MSCs) have been identified as an attractive cell population in regenerative medicine. In view of future therapeutic applications, the study of specific differentiation‐related gene expression is a pivotal prerequisite to define the most appropriate MSC source for clinical translation. In this context, it is crucial to use stable housekeeping genes (HGs) for normalization of qRT‐PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HGs comparing MSCs from different sources under various differentiation conditions is still missing. In this pivotal study, we compared the expression levels of 12 genes (ACTB, Β2M, EF1alpha, GAPDH, GUSB, PPIA, RPL13A, RPLP0, TBP, UBC, YWHAZ and 18S rRNA) to assess their suitability as HGs in MSCs during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HGs including 18S rRNA, B2M and ACTB were inadequate for normalization, whereas TBP/YWHAZ/GUSB were frequently identified among the best performers. Moreover, we showed the dramatic effects of suboptimal HGs choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSCs. Thus, in the emerging field of regenerative medicine, the identification of the most appropriate MSC source and cell line is so crucial for the treatment of patients that being inaccurate in the first step of the stem cell characterization can bring important consequences for the patients and for the promising potential of stem cell therapy.     

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (<i>Paeonia lactiflora</i> Pall.) in Response to Drought Stress

Herbaceous peony (Paeonia lactiflora Pall.), as a high-end cut flower in the international market, has high ornamental and medicinal values. But in Northern China, drought is a major environmental factor influencing the growth and development of P. lactiflora. Quantitative real-time polymerase chain reaction (qRT-PCR) can evaluate gene expression levels under different stress conditions, and stable internal reference is the key for qRT-PCR. At present, there is no systematic screening of internal reference for correcting gene expressions of P. lactiflora in response to drought stress. In this study, 10 candidate genes [ubiquitin (UBQ2), UBQ1, elongation factor 1-α (EF-1α), Histidine (His), eukaryotic initiation factor (eIF), tubulin (TUB), actin (ACT), UBQ3, ACT2, RNA polymerase II (RNA Pol II)] were chosen, and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress. Due to the difference of operation methods, the results of different analysis were distinct, and the final comprehensive analysis indicated that EF-1α was a relatively stable internal reference gene for P. lactiflora under drought stress. Also, UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis. This study will lay a foundation for screening the key genes of P. lactiflora in response to drought stress.     

Temperature and Development Impacts on Housekeeping Gene Expression in Cowpea Aphid,Aphis craccivora (Hemiptera: Aphidiae)

Quantitative real-time PCR (qRT-PCR) is a powerful technique to quantify gene expression. To standardize gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to consistently expressed housekeeping genes (HKGs) is required. In this study, ten candidate HKGs including elongation factor 1 α (EF1A), ribosomal protein L11 (RPL11), ribosomal protein L14 (RPL14), ribosomal protein S8 (RPS8), ribosomal protein S23 (RPS23), NADH-ubiquinone oxidoreductase (NADH), vacuolar-type H+-ATPase (ATPase), heat shock protein 70 (HSP70), 18S ribosomal RNA (18S), and 12S ribosomal RNA (12S) from the cowpea aphid, Aphis craccivora Koch were selected. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔC_t method were employed to evaluate the expression profiles of these HKGs as endogenous controls across different developmental stages and temperature regimes. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidate HKGs, RPS8, RPL14, and RPL11 were the three most stable HKGs across different developmental stages and temperature conditions. This study is the first step to establish a standardized qRT-PCR analysis in A. craccivora following the MIQE guideline. Results from this study lay a foundation for the genomics and functional genomics research in this sap-sucking insect pest with substantial economic impact.     

Evaluation of endogenous reference genes for analysis of gene expression with real-time RT-PCR during planarian regeneration

It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell types, developmental stages, and/or sample treatment. To select the most stable endogenous reference genes during planarian regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder software. Expression levels of the Djsix-1 gene were studied in parallel with ACTB and DjEF2 both or each and 18S rRNA as reference during regeneration. The results showed that ACTB was the most stable expressed reference gene in the planarian regeneration.     

Reference Gene Selection for Real-Time Quantitative PCR Analysis of the Mouse Uterus in the Peri-Implantation Period

The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm^PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.     

Identification of optimal housekeeping genes for examination of gene expression in bovine corpus luteum

The selection of proper housekeeping genes for studies requiring genes expression normalization is an important step in the appropriate interpretation of results. The expression of housekeeping genes is regulated by many factors including age, gender, type of tissue or disease. The aim of the study was to identify optimal housekeeping genes in the corpus luteum obtained from cyclic or pregnant cows. The mRNA expression of thirteen housekeeping genes: C2orf29, SUZ12, TBP, TUBB2B, ZNF131, HPRT1, 18s RNA, GAPDH, SF3A1, SDHA, MRPL12, B2M and ACTB was measured by Real-time PCR. Range of cycle threshold (C_t) values of the tested genes varied between 12 and 30 cycles, and 18s RNA had the highest coefficient of variation, whereas C2orf29 had the smallest coefficient. GeNorm software demonstrated C2orf29 and TBP as the most stable and 18s RNA and B2M as the most unstable housekeeping genes. Using the proposed cut-off value (0.15), no more than two of the best GeNorm housekeeping genes are proposed to be used in studies requiring gene expression normalization. NormFinder software demonstrated C2orf29 and SUZ12 as the best and 18s RNA and B2M as the worst housekeeping genes. The study indicates that selection of housekeeping genes may essentially affect the quality of the gene expression results.     

Selection of Suitable Housekeeping Genes for Real-Time Quantitative PCR in CD4+ Lymphocytes from Asthmatics with or without Depression

Objective

No optimal housekeeping genes (HKGs) have been identified for CD4⁺ T cells from non-depressive asthmatic and depressive asthmatic adults for normalizing quantitative real-time PCR (qPCR) assays. The aim of present study was to select appropriate HKGs for gene expression analysis in purified CD4⁺ T cells from these asthmatics.

Methods

Three groups of subjects (Non-depressive asthmatic, NDA, n = 10, Depressive asthmatic, DA, n = 11, and Healthy control, HC, n = 10 respectively) were studied. qPCR for 9 potential HKGs, namely RNA, 28S ribosomal 1 (RN28S1), ribosomal protein, large, P0 (RPLP0), actin, beta (ACTB), cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK1), beta-2-microglobulin (B2M), glucuronidase, beta (GUSB) and ribosomal protein L13a (RPL13A), was performed. Then the data were analyzed with three different applications namely BestKeeper, geNorm, and NormFinder.

Results

The analysis of gene expression data identified B2M and RPLP0 as the most stable reference genes and showed that the level of PPIA was significantly different among subjects of three groups when the two best HKGs identified were applied. Post-hoc analysis by Student-Newman-Keuls correction shows that depressive asthmatics and non-depressive asthmatics exhibited lower expression level of PPIA than healthy controls (p<0.05).

Conclusions

B2M and RPLP0 were identified as the most optimal HKGs in gene expression studies involving human blood CD4⁺ T cells derived from normal, depressive asthmatics and non-depressive asthmatics. The suitability of using the PPIA gene as the HKG for such studies was questioned due to its low expression in asthmatics.