Selection of suitable reference genes for quantitative real-time PCR in trabecular meshwork cells under oxidative stress

Oxidative stress-induced dysfunction in trabecular meshwork (TM) cells is considered a major alteration that can lead to glaucoma. Hydrogen peroxide (H₂O₂) is the most widely used agent for inducing oxidation in TM cells in vitro. Quantitative real-time PCR (qPCR) is an important method for studying alterations in gene expression, and suitable (i.e. invariant) reference genes must be defined to normalize expression levels. In this study, eight common reference genes, i.e. PRS18, ACTB, B2M, GAPDH, PPIA, HPRT1, YWHAZ, and TBP, were evaluated for use in studies of H₂O₂-induced dysfunction in TM cells. Three established algorithms, geNorm, NormFinder, and BestKeeper, were used to analyze the reference genes. ACTB expression was least affected by H₂O₂ treatment in TM cells, and the combination of PPIA and HPRT1 was the most suitable gene pair for normalization. GAPDH and TBP were the most unstable genes and accordingly should be avoided in experiments with TM cells. These results provide a foundation for analyses of the mechanisms underlying glaucoma, and emphasize the importance of selecting suitable reference genes for qPCR studies.     

Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.     

Selection and evaluation of RT-qPCR reference genes for expression analysis in the tiny egg parasitoid wasp,Trichogramma dendrolimi matsumura (Hymenoptera: Trichogrammatidae)

The egg parasitoid, Trichogramma spp., is an important biological control agent used against a broad range of Lepidopteran pests in agriculture and forestry. The biology of Trichogramma has been studied in details. Further studies should focus on the molecular mechanisms of Trichogramma by qualifying the expression of related genes It is critical to select appropriate reference genes for normalizing RT-qPCR results and establishing a robust method for quantifying target gene expression. This study aimed to identify and validate appropriate reference genes for use in RT-qPCR analysis of Trichogramma dendrolimi. Ten candidate housekeeping genes, namely beta-actin (ACTIN), forkhead box O (FOXO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (HSP90), ribosomal protein L10a (RPL10a), L18 (RPL18), L28 (RPL28), S13 (RPS13), S15 (RPS15), and superoxide dismutase (SOD), were tested for their suitability as reference genes for developmental stage (3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th day after parasitization), tissue (head, thorax, and abdomen of adults), sex of adults (male and female), and temperature (17℃, 25℃, and 32℃). According to the GeNorm analysis, a robust analysis should involve using an appropriate combination of reference genes, namely, at least three genes for different development stages, two genes for different tissues, two genes for different sex, and two genes for different temperatures, respectively. According to the RelFinder method by the integrated results of GeNorm, NormFinder, BestKeeper, and the ΔCt method, we identified the developmental stage-specific reference genes SOD, GAPDH, and ACTIN; tissue-specific reference genes RPL18 and RPS15; sex-specific reference genes RPL18 and SOD; and temperature-specific reference genes RPL18 and RPL10a. This study provides a standardized procedure for the quantification of gene expression in T. dendrolimi and will be helpful for future biological control programs using Trichogramma wasps.     

<Emphasis Type="Italic">RPN1</Emphasis>, a new reference gene for quantitative data normalization in lung and kidney cancer

Quantitative methods of gene expression analysis in tumors require accurate data normalization, which allows comparison of different specimens with unknown mRNA/cDNA concentrations. For this purpose, reference genes with stable expression are used (e.g., GAPDH, ACTB, HPRT1, or TBP). The problem of choosing proper reference genes is still a topical issue, because well-known reference genes can be unsuitable for certain cancer types and their inappropriate use without additional testing can lead to wrong conclusions. A recently developed bioinformatical approach was employed to identify a new potential reference gene for lung and kidney tumors, RPN1, located on the long arm of chromosome 3. The method employed the mining of the dbEST and Oncomine databases and functional analysis of genes. RPN1 was selected from approximately 1500 candidate housekeeping genes. Using comparative genomic hybridization with NotI microarrays, we found no methylation, deletions, and/or amplifications in the RPN1-containing locus in 56 nonsmall cell lung and 42 clear cell renal cell cancer specimens. Real-time PCR showed that variation of RPN1 mRNA levels in nonsmall cell lung cancer and clear-cell renal cancer was low and comparable to that of the known reference genes GAPDH and GUSB, respectively. Expression levels of two hyalouronidase genes, HYAL1 and HYAL2, were assessed using the suggested references gene pairs (RPN1-GAPDH for lung cancer and RPN1-GUSB for kidney cancer), and these combinations were shown to produce accurate and reproducible data. These results suggest that RPN1 is a new, promising reference gene for quantitative data normalization in gene expression studies for lung and kidney cancers.     

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

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 C_q, 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.     

Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae)

To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.     

Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR

Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.     

Selection of Stable Reference Genes for Quantitative Real-Time PCR on <i>Paeonia ostii</i> T. Hong et J. X. Zhang Leaves Exposed to Different Drought Stress Conditions

The definition of relatively stable expressed internal reference genes is essential in both traditional blotting quantification and as a modern data quantitative strategy. Appropriate internal reference genes can accurately standardize the expression abundance of target genes to avoid serious experimental errors. In this study, the expression profiles of ten candidate genes, ACT1, ACT2, GAPDH, eIF1, eIF2, α-TUB, β-TUB, TBP, RNA Pol II and RP II, were calculated for a suitable reference gene selection in Paeonia ostii T. Hong et J. X. Zhang leaves under various drought stress conditions. Data were processed by the four regularly used evaluation software. A comprehensive analysis revealed that RNA Pol II was the most stable gene and eIF2 was the least stable one. In addition, the geNorm program provided the optimal choice of two reference gene combination, RNA Pol II and β-TUB, for qRT-PCR normalization in P. ostii subjected to different drought stress levels. Our research provided convenience for gene expression analysis in P. ostii under drought stress and promoted research of effective methods to alleviate P. ostii drought stress in the future.     

Selection of optimal reference genes for expression analysis in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> during development,under changing nutrient conditions,and after exposure to abiotic stresses

The selection of suitable reference genes is crucial for accurate quantification of gene expression. To identify suitable reference genes in Beauveria bassiana, the expression of 14 candidates (18S, 28S, β-Tub, GAPD, γ-Act, TEF, HGPT, His3, His2A, TBP, CypA, CypB, PP1, and CrzA) was measured by quantitative polymerase chain reaction at different development stages and under various nutritional and stress conditions. Expression stability, as evaluated by the geNorm and NormFinder programs, revealed that His2A/γ-Act/CrzA was the most stably expressed set of genes throughout development, while 28S/PP1/CypA and His2A/γ-Act/CypA were the most stably expressed gene sets under a variety of nutritional and stress conditions, respectively. Overall, the most stably expressed genes under all conditions examined were PP1, γ-Act, and CypA.