Reference Genes For RT-qPCR Normalization Research Articles

Selection and validation of reference genes for RT-qPCR normalization of porcine alveolar macrophages (PAMs) for PRRSV studies

Porcine alveolar macrophages (PAMs) are widely used for in vitro studies of porcine respiratory viruses. Gene expression in these cells is altered by viral infection and cellular immune response. Real-time reverse transcription polymerase chain reaction (RT-qPCR) is a powerful technique for analyzing these changes. In order to obtain reliable quantitative RT-qPCR data and come to sound conclusions, stable reference genes are needed for normalization of target gene expression. In the present study, we evaluated the expression stability of nine reference genes in PAMs during cultivation and upon porcine reproductive and respiratory syndrome virus (PRRSV) inoculation. Using geNorm and NormFinder algorithms, we identified PSAP and GAPDH as the most stable reference genes under all experimental conditions. The selected reference genes were used for the normalization of CD163 expression under different conditions. This study demonstrates that selection of appropriate reference genes is essential for normalization and validation of RT-qPCR data across all experimental conditions. This study provides a new set of stable reference genes for future studies with porcine respiratory viruses in PAMs.

Selection and validation of endogenous reference genes for RT-qPCR normalization in different stresses and tissues of the tiger milk mushroom, Pleurotus tuber-regium.

Pleurotus tuber-regium is a sclerotium-producing mushroom with pharmacological and nutritional value. Many researches applied molecular biological strategies that allow analyzing numerous genes expressed in response to different growth stresses or tissues. In order to capture accurate and reliable results in gene expression studies, it is necessary to select appropriate internal reference genes. They must have relatively uniform expression in the majority of tested samples, thereby reducing the impact of the sample itself on the RT-qPCR results. The selection and validation of 14 candidate reference genes, including α-tub, β-tub, γ-tub, GAPDH, Tif-5a, Tef-1α, ATPase, acyl-CoA, U2ribo, S/G, Wlp, VtpAsE, E3upl, and Sdh, were carried out for gene expression analysis in P. tuber-regium mycelia in response to different temperatures, drought levels, and salinity shifts, fruitbody, and sclerotium. Four statistical algorithms, NormFinder, geNorm, BestKeeper, and RefFinder, were recruited to evaluate the transcription stability of candidate reference genes. The RT-qPCR expression stability analysis indicated that the E3upl and Tif-5a were the most stable expressed genes among all experimental samples, so the combination of these two reference genes is suitable for the analysis of gene expression patterns in P. tuber-regium.

Selection and optimization of reference genes for RT-qPCR normalization: A case study in Solanum lycopersicum exposed to UV-B

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum, there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT+TUB as the best pair of RGs for leaves, PP2A+GAPDH+TRX h1 for stems and TUB+UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation.

Selection and validation of reference genes for RT-qPCR normalization in different tissues of milk thistle (Silybum marianum, Gaert.).

Quantitative reverse transcription PCR is a sensitive technique for evaluating transcriptional profiles in different experimental datasets. To obtain a reliable quantification of the transcripts level, data normalization with stable reference genes is required. Stable reference genes are identified after analysis of their transcripts profile in every new experiment and species of interest. In Silybum marianum, a widely cultivated officinal plant, only few gene expression studies exist, and reference genes for RT-qPCR studies in the diverse plant tissues have never been investigated before. In this work, the expression stability of 10 candidate reference genes was evaluated in leaves, roots, stems and fruits of S. marianum grown under physiological environmental condition. The stability values for each candidate reference gene were calculated by four canonical statistical algorithms GeNorm, NormFinder, Bestkeeper and ΔCt method in different subsets of samples, then they were ranked with RefFinder from the most to the least suitable for normalization. Best combinations of reference genes are finally proposed for different experimental data sets, including all tissues, vegetative, and reproductive tissues separately. Three target genes putatively involved in important biosynthetic pathway leading to key metabolites in the fruits of milk thistle, such as silymarin and fatty acids, were analyzed with the chosen panels of reference genes, in comparison to the ones used in previous papers. To the best of our knowledge, this is the first report on a reliable and systematic identification and validation of the reference genes for RT-qPCR normalization to study gene expression in S. marianum.

Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis in tamarillo (Solanum betaceum Cav.)

Quantitative reverse transcriptase PCR is a powerful tool for precise gene expression quantification. Using suitable reference genes is crucial for a proper normalization and interpretation of results, particularly in dynamic transcriptional networks with the involvement of several miRNAs and their targets, such as somatic embryogenesis (SE). Here, we aimed to identify genes to be used as internal controls for quantitative detection of miRNAs and mRNAs during the SE process in Solanum betaceum. The expression of EF1α, GAPDH, ACT, UBQ10, Fe-SOD, U6, snoR14, miR159a, miR162 and miR166a was tested in nine time-points of the SE process, particularly during induction, embryo development and conversion. Analyses of transcriptional stability were performed using RefFinder tool that integrates geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among these candidates, Fe-SOD, ACT and U6 emerged consistently as the most suitable to normalize gene expression, whereas miR166a was the most appropriate for normalization in calli samples. In order to validate these results, expression of miR396 and GRF1 was quantified and normalized with the most and the least stable genes and also with the most stable miRNA. Fe-SOD + ACT revealed to be the optimal combination for precise quantification of miRNA-mediated regulation of gene expression during SE in tamarillo. Fe-SOD + ACT is the most suitable combination of reference genes for RT-qPCR normalization and accurate quantification of miRNAs and their targets during tamarillo somatic embryogenesis induction, embryo development and conversion stages.

Assessment of Suitable Reference Genes for RT-qPCR Normalization with Developmental Samples in Pacific Abalone Haliotis discus hannai

Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.

Reference genes for accurate evaluation of expression levels in Trichophyton interdigitale grown under different carbon sources, pH levels and phosphate levels

Tinea pedis is a type of dermatophytosis caused by anthropophilic keratinolytic fungi such as Trichophyton interdigitale. Quantitative reverse transcription PCR (RT-qPCR) is a reliable and reproducible technique for measuring changes in target gene expression across various biological conditions. A crucial aspect of accurate normalization is the choice of appropriate internal controls. To identify reference genes for accurate evaluation of expression levels in T. interdigitale, the transcription levels of eight candidate reference genes (adp-rf, β-act, ef1-α, gapdh, psm1, sdha, rpl2 and ubc) and one target gene (Tri m4) were analysed by RT-qPCR after growing the dermatophyte under different environmental conditions. The results obtained from expression stability evaluations with NormFinder, geNorm, BestKeeper, and RefFinder software demonstrated that adp-rf and psm1 were the most stable internal control genes across all experimental conditions. The present study constitutes the first report of the identification and validation of reference genes for RT-qPCR normalization for T. interdigitale grown under different environmental conditions resembling the conditions encountered by fungi during invasion of skin.

Genome-wide identification of new reference genes for RT-qPCR normalization in CGMMV-infected Lagenaria siceraria.

Lagenaria siceraria is an economically important cucurbitaceous crop, but suitable reference genes (RGs) to use when the plants are infected by cucumber green mottle mosaic virus (CGMMV) have not been determined. Sixteen candidate RGs of both leaf and fruit and 18 candidate RGs mostly from separate RNA-Seq datasets of bottle gourd leaf or fruit were screened and assessed by RT-qPCR. The expression stability of these genes was determined and ranked using geNorm, NormFinder, BestKeeper and RefFinder. Comprehensive analysis resulted in the selection of LsCYP, LsH3, and LsTBP as the optimal RGs for bottle gourd leaves, and LsP4H, LsADP, and LsTBP for fruits. LsWD, LsGAPDH, and LsH3 were optimal for use in both leaves and fruits under the infection of CGMMV. Isopentenyl transferase (IPT) and DNA-directed RNA polymerase (DdRP) were used to validate the applicability of the most stable identified RGs from bottle gourd in response to CGMMV. All the candidate RGs performed in RT-qPCR consistently with the data from the transcriptome database. The results demonstrated that LsWD, LsGAPDH and LsH3 were the most suitable internal RGs for the leaf, and LsH3, LsGAPDH, LsP4H and LsCYP for the fruit.

Selection and Evaluation of Appropriate Reference Genes for RT-qPCR Normalization of Volvariella volvacea Gene Expression under Different Conditions.

Volvariella volvacea (V. volvacea), commonly referred to as Chinese (paddy straw) mushroom, is a basidiomycete with a protein-rich volva and pileus. Selecting appropriate reference genes is a crucial step in the normalization of quantitative real-time PCR data. Therefore, 12 candidate reference genes were selected from the V. volvacea transcriptome based on previous studies and then BestKeeper, geNorm, and NormFinder were used to identify reference genes stably expressed during different developmental stages and conditions. Of the 12 candidate reference genes, SPRY domain protein (SPRYp), alpha-tubulin (TUBα), cyclophilin (CYP), L-asparaginase (L-asp), and MSF1-domain-containing protein (MSF1) were the most stably expressed under different experimental conditions, while 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), and beta-actin (ACTB) were the least stably expressed. This investigation not only revealed potential factors influencing the suitability of reference genes, but also identified optimal reference genes from a pool of candidate genes under a wide range of conditions.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a.

The powerful Quantitative real-time PCR (RT-qPCR) was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. Oenococcus oeni (O. oeni), as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB) species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes (proC, dnaG, rpoA, ldhD, ddlA, rrs, gyrA, gyrB, and dpoIII) were analyzed to determine the most stable reference genes for RT-qPCR in O. oeni SD-2a under different ethanol stress conditions (8, 12, and 16% (v/v) ethanol). The transcript stabilities of these genes were evaluated using the algorithms geNorm, NormFinder, and BestKeeper. The results showed that dnaG and dpoIII were selected as the best reference genes across all experimental ethanol conditions. Considering single stress experimental modes, dpoIII and dnaG would be suitable to normalize expression level for 8% ethanol shock treatment, while the combination of gyrA, gyrB, and rrs would be suitable for 12% ethanol shock treatment. proC and gyrB revealed the most stable expression in 16% ethanol shock treatment. This study selected and validated for the first time the reference genes for RT-qPCR normalization in O. oeni SD-2a under ethanol stress conditions.

Optimal reference genes for RT-qPCR normalization in the newborn

It is difficult to identify reliable reference genes for transcriptomic analyses in biofluids such as saliva. This situation is particularly relevant for the newborn population, where rapid development is associated with dynamic changes in gene expression. Real-time gene expression monitoring holds great promise for elucidating disrupted pathways that result in morbidities unique to this population, such as retinopathy of prematurity, but its impact depends on identifying stable and consistently expressed genes across a wide range of gestational ages. We extracted total RNA from 400 neonatal saliva samples (postconceptional ages: 32 5/7 to 48 2/7 weeks), converted it to cDNA, and pre-amplified and analyzed it by qPCR for three commonly used reference genes, ACTB, GAPDH, and YWHAZ. Relative quantification was determined using the Δ Ct method. Data were analyzed as a whole and also stratified by age and sex. Descriptive statistics and homogeneity of variance were performed to identify optimal reference genes. Data analyzed from all ages and both sexes showed significant expression variation for ACTB, while GAPDH and YWHAZ showed greater stability. Male infants exhibited increased expression variation compared to females for ACTB, but neither GAPDH nor YWHAZ showed significant variance for either sex. We suggest that ACTB is an unreliable reference gene for the newborn population. Males showed significantly more variation in ACTB expression compared to females, which suggests a sex-specific developmental role for this biomarker. By contrast, GAPDH and YWHAZ were less variable and therefore preferable for use in neonates. Our findings may improve the use of reference genes for the RT-qPCR platform in the newborn over a wide range of gestational ages, thereby minimizing the likelihood of erroneous interpretation of gene expression during rapid growth, development, and differentiation.

Selection of Suitable Reference Genes for RT-qPCR Normalization under Abiotic Stresses and Hormone Stimulation in Persimmon (Diospyros kaki Thunb).

The success of quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to quantify gene expression depends on the stability of the reference genes used for data normalization. To date, systematic screening for reference genes in persimmon (Diospyros kaki Thunb) has never been reported. In this study, 13 candidate reference genes were cloned from 'Nantongxiaofangshi' using information available in the transcriptome database. Their expression stability was assessed by geNorm and NormFinder algorithms under abiotic stress and hormone stimulation. Our results showed that the most suitable reference genes across all samples were UBC and GAPDH, and not the commonly used persimmon reference gene ACT. In addition, UBC combined with RPII or TUA were found to be appropriate for the "abiotic stress" group and α-TUB combined with PP2A were found to be appropriate for the "hormone stimuli" group. For further validation, the transcript level of the DkDREB2C homologue under heat stress was studied with the selected genes (CYP, GAPDH, TUA, UBC, α-TUB, and EF1-α). The results suggested that it is necessary to choose appropriate reference genes according to the test materials or experimental conditions. Our study will be useful for future studies on gene expression in persimmon.

Validation of reference genes for RT-qPCR normalization in Iris. lactea var. chinensis leaves under different experimental conditions

Iris. lactea var. chinensis (I. lactea var. chinensis) is highly tolerant of Cu, Pb, drought, NaCl and cold. To study the molecular mechanism of I. lactea var. chinensis under these stresses, it is necessary to select reliable reference genes for RT-qPCR study. In the present study, the expression stability of nine candidate reference genes, namely, beta-tubulin (TUBLIN6), ACTIN11, translation initiation factor 4A-1 (EIF4A1), cytosolic phosphoglycerate kinase (PGK), TIP41-like family protein (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAP), phosphatase 2A (PP2Acs), cyclophilin (CYP) and ubiquitin (UBQ), was assessed in I. lactea var. chinensis plants subjected to five abiotic stresses using GeNorm and NormFinder. A combination of GAP, ACTIN11, TIP41, EIF4A1 and PGK was identified as appropriate genes for normalization under PEG stress, whereas the combination of UBQ, PGK, TIP41 and PP2Acs was the most suitable under NaCl stress. PP2Acs, ACTIN11 and UBQ exhibited the most stable expression under Pb stress. For Cu-treated leaves, CYP, GAP and TIP41 were the most stably expressed, while GAP, PP2Acs and TIP41 for cold-treated leaves. Generally, the analyses found that TIP41, CYP, PGK, GAP and PP2Acs were the most stable genes under five abiotic stress conditions. These results would contribute the more accurate and widespread use of RT-qPCR in I. lactea var. chinensis gene analysis.

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.