Reference Genes For Expression Analysis Research Articles

Transcriptome-guided selection of stable reference genes for expression analysis in spinach.

Accurate measurement of gene expression levels is vital for advancing plant biology research. This study explores the identification and validation of stable reference genes (RGs) for gene expression analysis in Spinacia oleracea. Leveraging transcriptome data from various developmental stages, we employed rigorous statistical analyses to identify potential RGs. A total of 1196 candidate genes were initially screened based on expression variability, with subsequent refinement using criteria such as low variance and stability. Among 12 commonly used candidate RGs, EF1α and H3 emerged as the most stable across diverse experimental conditions, while GRP and PPR exhibited lower stability. These findings were further validated through qRT-PCR assays and comprehensive statistical analyses, including geNorm, NormFinder, BestKeeper, and RefFinder. Our study underscores the importance of systematic RG selection to ensure accurate normalization in gene expression studies, particularly in the context of S. oleracea developmental stages and physiological processes like flowering. These validated RGs provide a robust foundation for future gene expression analysis in S. oleracea and contribute to the advancement of molecular research in plant biology.

Identification and selection of reference genes for analysis of gene expression by quantitative real-time PCR in the euhalophyte Suaeda altissima (L.) Pall.

ABSTRACT Сoding sequences of seven housekeeping genes: actin SaACT7, ubiquitin-conjugating protein SaUBC10, glyceraldehyde-3-phosphate dehydrogenase SaGAPDH, protein of the large subunit of ribosomes SaL2, α-tubulin SaTUA, translation elongation factor SaeEF1α, and protein phosphatase SaPP2A were identified as candidate reference genes for expression analysis of target genes in the extremely salt tolerant plant Suaeda altissima (L.) Pall. The expression profiles of the genes differed. SaACT7 and SaeEF1α demonstrated the highest expression levels, while the lowest expression levels were found for SaPP2A and SaTUA. SaPP2A and SaeEF1α genes were the most stably expressed at different steady-state salinity levels and different nitrate concentrations in nutrient solutions (NSs). SaL2, SaPP2A, and SaeEF1α genes showed the greatest stability of expression when nitrate was added to nutrient solution of plants grown under conditions of nitrate deficiency. Less constant expression was demonstrated in this experiment by SaACT7 and SaTUA. SaL2, SaACT7, SaeEF1α, and SaUBC10 genes showed the smallest expression changes under salt shock. To validate the use of the most stably expressed genes for normalization of gene expression, we checked them as reference genes to study the expression of the nitrate transporter gene SaNPF6.3 in S. altissima roots under conditions of different salinity and different nitrate supply.

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus.

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.

Validation of the Reference Genes for Expression Analysis in the Hippocampus after Transient Ischemia/Reperfusion Injury in Gerbil Brain.

Transient brain ischemia in gerbils is a common model to study the mechanisms of neuronal changes in the hippocampus. In cornu ammonnis 2-3, dentate gyrus (CA2-3,DG) regions of the hippocampus, neurons are resistant to 5-min ischemia/reperfusion (I/R) insult, while cornu ammonnis 1 (CA1) is found to be I/R-vulnerable. The quantitative polymerase chain reaction (qRT-PCR) is widely used to study the expression of genes involved in these phenomena. It requires stable and reliable genes for normalization, which is crucial for comparable and reproducible analyses of expression changes of the genes of interest. The aim of this study was to determine the best housekeeping gene for the I/R gerbil model in two parts of the hippocampus in controls and at 3, 48, and 72 h after recanalization. We selected and tested six reference genes frequently used in central nervous system studies: Gapdh, Actb, 18S rRNA, Hprt1, Hmbs, Ywhaz, and additionally Bud23, using RefFinder, a comprehensive tool based on four commonly used algorithms: delta cycle threshold (Ct), BestKeeper, NormFinder, and geNorm, while Hprt1 and Hmbs were the most stable ones in CA2-3,DG. Hmbs was the most stable in the whole hippocampal formation. This indicates that the general use of Hmbs, especially in combination with Gapdh, a highly expressed reference gene, seems to be suitable for qRT-PCR normalization in all hippocampal regions in this model.

Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress.

Reference genes, also referred to as housekeeping genes (HKGs), play an important role in gene expression analysis by serving as an internal control. These HKGs are usually involved in basic cellular functions and their expression should remain at relatively constant levels. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been used to measure gene expression. Since the normalization of gene expression data depends on baseline expression of HKGs, it is important to identify and verify true HKGs for the qRT-PCR analysis. The goal of this study is to identify and confirm HKGs in Bradyrhizobium diazoefficiens, a nitrogen fixing bacterium which forms a symbiotic relationship with soybean. By revealing such HKGs, the normalization of gene expression would be more robust, reliable, and consistent. Here, we analyzed previous gene expression data for B. diazoefficiens under multiple environmental conditions. As a result, we identified seven constitutively expressed genes among 8453 genes across all conditions. Their fold-change values were within a range of −1.25-fold < x < 1.25-fold. We adopted GeNorm, NormFinder, and comparative ∆Ct methods to rank the seven candidate genes based on their expression stability. To validate these potential HKGs, we measured their expression in various experimental conditions, such as heat, pH, and heavy metal stress. The HKGs that were found in B. diazoefficiens were also applied in closely related species by identifying their homologs.

Selection of suitable reference gene for gene expression studies during groundnut seed germination

Understanding the mechanism of seed germination requires a sturdy selection of reference genes for expression analysis using qRT-PCR. The accurate normalization of genes becomes necessary to circumvent erroneous result, as housekeeping genes does not remain stable over the different experimental condition in various tissue or species. Reliable and stable reference gene during ethrel induced germination of groundnut seeds is not reported yet. In this study, seven candidate reference genes were selected based on previous reports in groundnut under different experimental conditions. Seeds of NRCG 14380 (dormant) and TAG 24 (non-dormant) genotypes were treated with ethrel and sampled at 0, 6, 12 and 24 hours after incubation. The stability of reference genes such as actin1 (ACT1), actin11 (ACT11), alcohol dehydrogenase (ADH3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), ubiquitin C (UBC) and α-Tubulin (TUA) was analyzed through a different statistical algorithm such as BestKeeper, NormFinder, geNorm and its consensus stability ranking was retrieved using RefFinder. Among all reference genes studied, ACT1 was found most stable reference gene in our study. Stability and reliability of ACT1 and ADH3 (most stable) reference genes were further validated through qRT- PCR with relative quantification of two germination related genes ACC oxidase1 (ACO1) and gibberellic acid regulated protein which showed consistency in their expression level. Our result revealed a stable reference gene that could be used as an internal control gene for gene expression study of ethrel treated groundnut seed germination.

Transcriptional search to identify and assess reference genes for expression analysis in Solanumlycopersicum under stress and hormone treatment conditions

Tomato (Solanumlycopersicum) is a model plant for research on fruit development and stress response, in which gene expression analysis is frequently conducted. Quantitative PCR (qPCR) is a widely used technique for gene expression analysis, and the selection of reference genes may affect the accuracy of results and even conclusions. Although there have been some frequently used reference genes in tomato, it has been shown that the expressions of some of these genes are not constant in different tissues and environmental conditions. Moreover, little information on genomic identification of reference genes is available in tomato. Here, we mined the publicly available transcriptional sequencing data and screened out fifteen candidate reference genes, and the expression stability of these candidate genes and seven traditionally used ones were evaluated under stress and hormone treatment. The results showed that over half of the selected candidate references were housekeeping genes in tomato cells. Among the candidate reference genes and the traditionally used ones, the most stably expressed genes varied under different treatments, and most of these genes were recommended as preferred reference genes at least once except Solyc04g009030 and Solyc07g066610, two traditionally used reference genes. This study provides some novel reference genes in tomato, and the preferred reference genes under different environmental stimuli, which may be useful for future research. Our study suggests that excavating stably expressed genes from transcriptome sequencing data is a reliable approach to screening reference genes for qPCR analysis.

Selection of optimal qRT-PCR reference genes for Aconitum vilmorinianum

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.

Identification of suitable mRNAs and microRNAs as reference genes for expression analyses in skin cells under sex hormone exposure.

Quantitative RT-PCR is the most accurate technique for the study of gene expression profiles, however, to ensure the accuracy of qPCR results, suitable reference genes are necessary for data normalization. Hormones influence the development and function of skin cells, regulating the expression of genes and miRNAs. Nevertheless, the stability of reference genes after sex hormone treatment has not been thoroughly investigated. In this study, we evaluated the expression of a set of candidate mRNAs and microRNsA (miRNA) as reference genes in keratinocytes (HaCaT cells), primary human fibroblasts and a melanoma cell line (LM-36 cells) under testosterone or 17β-estradiol treatment. Two algorithms, namely geNorm, Best-Keeper, and the comparative ΔCt method were used to evaluate the expression stability of the candidate reference genes. The comprehensive ranking showed that TBP and miR-191-5p are the most stable expressed genes across all cultured cells under hormone treatment. Furthermore, we observed that GAPDH, HPRT1 and U6 snRNA expression may be altered by hormone exposure, thus, these genes are not recommended as reference genes. In conclusion, the present study provides, to the best of our knowledge, the first evaluation of expressed mRNA(s) and miRNA(s) as reference genes in three different types of skin cells under the stimulation of sex hormones.

Validation of reference genes for expression analysis in a murine trauma model combining traumatic brain injury and femoral fracture

Systemic and local posttraumatic responses are often monitored on mRNA expression level using quantitative real-time PCR (qRT-PCR), which requires normalisation to adjust for confounding sources of variability. Normalisation requests reference (housekeeping) genes stable throughout time and divergent experimental conditions in the tissue of interest, which are crucial for a reliable and reproducible gene expression analysis. Although previous animal studies analysed reference genes following isolated trauma, this multiple-trauma gene expression analysis provides a notable study analysing reference genes in primarily affected (i.e. bone/fracture callus and hypothalamus) and secondarily affected organs (i.e. white adipose tissue, liver, muscle and spleen), following experimental long bone fracture and traumatic brain injury. We considered tissue-specific and commonly used top-ranked reference candidates from different functional groups that were evaluated applying the established expression stability analysis tools NormFinder, GeNorm, BestKeeper and RefFinder. In conclusion, reference gene expression in primary organs is highly time point as well as tissue-specific, and therefore requires careful evaluation for qRT-PCR analysis. Furthermore, the general application of Ppia, particularly in combination with a second reference gene, is strongly recommended for the analysis of systemic effects in the case of indirect trauma affecting secondary organs through local and systemic pathophysiological responses.

Susceptibility of mountain pine beetle (Dendroctonus ponderosae Hopkins) to gene silencing through RNAi provides potential as a novel management tool

The mountain pine beetle (MPB), Dendroctonus ponderosae, is an eruptive endemic forest pest that is undergoing substantial range expansion in response to recent climatic changes, breaching geographic barriers, exploiting novel hosts, and affecting millions of hectares of conifer forests in western North America. Current management approaches have been unable to keep pace with MPB population outbreaks, and novel and aggressive management responses are required as MPB’s range expansion progresses. Gene silencing through RNA interference (RNAi) is an emerging pest management approach that is being developed for agricultural pests, and has also been shown to be effective against some xylophagous forest pests, including the southern pine beetle (SPB), D. frontalis. When essential genes are targeted, RNAi can cause rapid insect mortality; here we focus on evaluating its effectiveness in MPB. We identified reference genes for quantitative real-time PCR (qPCR) and validated RNAi responses in MPB by analyzing gene expression and beetle survival. Using an adult bioassay that combined oral ingestion and dermal absorption of dsRNAs targeting three genes (hsp, iap, and shi), we measure gene expression and demonstrate silencing, as well as insect mortality, following dsRNA exposure. All three genes were silenced and all treatment beetles died within 7 d. This validates reference genes for expression analyses and demonstrates that MPB, similar to the congeneric SPB, has a highly sensitive RNAi response. Additionally, we document sex-specific differences in gene expression for one of the three target genes, hsp; any differences in gene expression and subsequent mortality based on sex must be considered as this technology progresses as a pest management tool. RNAi causes rapid insect mortality when essential genes are targeted, is highly specific to the target pest, and has no environmental contamination risks, making it an attractive approach for further development in forest pest suppression.

Identification of reference genes for transcript normalization in various tissue types and seedlings subjected to different abiotic stresses of woodland strawberry Fragaria vesca

Quantitative real-time PCR (qRT-PCR) is one of the most popular methods for gene expression studies. However, data on the usage of reference genes in strawberry are limited. In this study, we assayed expression dynamics of 20 candidate reference genes across various organs (root, leaf and flower), different developmental stages of fruits, and in seedlings under abiotic stresses (heat, cold, drought and salt) in woodland strawberry Fragaria vesca. Expression stability of those genes was analyzed by four commonly used statistical algorithms (geNorm, NormFinder, BestKeeper and dalteCt) for comparisons and rankings. Our results indicate that the expression stability of reference genes varies according to tissue types and experimental conditions. Furthermore, two reference genes are sufficient for normalization in tissues and seedlings under the four stress conditions in Fragaria vesca. We recommend FveSAND and FveMT1, FveCHC1 and FveGAPDH as reference genes in organs and fruits, FveCHC1 and FveTIM1, FveSAND and FveTIM1, FveCHP1 and FveAP1, FveCHC1 and FveEF 1a under heat, cold, drought and salt stress conditions, respectively. Our study provides a set of reliable standard reference genes for expression analyses of genes in various tissue types and seedlings under different environment conditions of Fragaria vesca.

Screening of reference genes for expression analysis in the study of soldier caste differentiation of Formosan subterranean termite Coptotermes formosanus Shiraki.

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a serious pest worldwide. Juvenile hormone analog (and its analogs such as methoprene) can induce the transformation of the worker caste into soldier caste in C. formosanus. However, several factors, such as feeding substrate and colony origin, influence the proportion of solider formation. The molecular mechanism of worker to soldier transformation of C. formosanus is still not clear. RT-qPCR is a powerful tool for molecular studies. Accurate gene quantification by the relative quantification method requires a stable expressed gene as the reference gene. However, no reference genes were available for this species in the methoprene bioassay. To study the problem of gene response to methoprene by RT-qPCR we have to first screen reference genes in C. formosanus. Workers were fed with methoprene. Termites were collected during the methoprene bioassay and separated into head and thorax+abdomen. Expression profiles of 10 candidate reference genes in the two body part types were investigated using RT-qPCR. The results were analyzed by a set of established methods (geNorm, NormFinder, BestKeeper, and RefFinder) as well as comparative ΔCt method. Our results suggest that RPS18 is the most stably-expressed gene both in the head and thorax+abdomen for expression analysis in the methoprene bioassay of C. formosanus. The screening of suitable reference genes in C. formosanus establishes the foundation for the molecular study of soldier caste differentiation in this species.

Selection of reference genes for expression analysis of plant-derived microRNAs in Plutella xylostella using qRT-PCR and ddPCR.

The establishment of an expression quantification system that can be easily applied for the comparison of microRNAs (miRNAs) from biological samples is an important step toward understanding functional mechanisms in organisms. However, there is lack of attention on the selection of reference genes for miRNA expression profiling in insect herbivores. Here, we explored the candidate reference genes in a notorious pest of cruciferous crops, Plutella xylostella, for normalization of miRNA expression in developmental stages and tissues and in response to a change of food source from artificial diet to host plant Arabidopsis thaliana. We first compared the expression levels and stability of eight small RNAs using qRT-PCR, and found that miR11 was the most suitable reference gene for expression quantification of the miRNAs. We then confirmed this finding using digital droplet PCR and further validated with a well-studied cross-kingdom miRNA derived from A. thaliana (ath-miR159a). However, none of the reference genes was applicable for all experimental conditions, and multiple reference genes were sometimes required within the same experiment. Our work provides a method for the selection of reference genes for quantification of plant-derived miRNAs, which paves the way for unveiling their roles in the insect-plant coevolution.

RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion.

Identification of atpD as an optimal reference gene to explore antibiotic resistance and stress tolerance in Rahnella aquatilis.

Rahnella aquatilis is a Gram-negative bacterial species with potential for agricultural and industrial applications, as well as a human pathogen. This study aims to identify an optimal reference gene to explore antibiotic resistance and stress tolerance in R. aquatilis using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Expression levels of 14 housekeeping genes in R. aquatilis were estimated by RT-qPCR under six different conditions: exponential phase, stationary phase, acid, salinity, antibiotic and oxidative stresses. BestKeeper and the ΔCt method were used to evaluate the stability of each gene. The atpD gene was stably expressed in all conditions, thus was selected and validated as an optimal reference gene. Transcript levels of 17 putative ampicillin-resistance genes in R. aquatilis strain HX2 were evaluated using the proposed RT-qPCR. Six genes encoding efflux transporters and β-lactamase were overexpressed after ampicillin treatment. Additionally, the expression of seven putative stress response genes in strain HX2 was assessed, and five genes were up-regulated by respective stress treatments. The atpD gene has been identified as an optimal reference gene for expression analysis of R. aquatilis responses to abiotic stresses by RT-qPCR. The proposed RT-qPCR is suitable for gene expression analysis in R. aquatilis, thus useful for studying antimicrobial resistance and stress tolerance in this bacterium and others closely related.

Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells

BackgroundThe proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45−/CD31−.MethodsWe evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software.ResultsThe results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes.ConclusionsApplying these findings for future gene expression analyses will help elucidate ASC biology.

Selection of Reference Genes for Expression Analysis in Chinese Medicinal Herb Huperzia serrata.

Huperzine A (HupA) is a powerful and selective inhibitor of acetylcholinesterase. It has attracted widespread attention endangering the ultimate plant sources of Lycopodiaceae family. In this study, we used Huperzia serrata, extensively used in Traditional Chinese medicine (TCM), a slow growing vascular plant as the model plant of the Lycopodiaceae family to develop and validate the reference genes. We aim to use gene expression platform to understand the gene expression of different tissues and developmental stages of this medicinal herb. Eight candidate reference genes were selected based on RNA-seq data and evaluated with qRT-PCR. The expression of L/ODC and cytochrome P450s genes known for their involvement in lycopodium alkaloid biosynthesis, were also studied to validate the selected reference genes. The most stable genes were TBP, GAPDH, and their combination (TBP + GAPDH). We report for the first time the reference gene of H. serrata’s different tissues which would provide important insights into understanding their biological functions comparing other Lycopodiaceae plants and facilitate a good biopharming approach.

Validation of reference genes for expression analysis in three Bupleurum species

Radix Bupleuri (root of Bupleurum spp.) is an important medicinal herb. Its lateral root number is one of the decisive factors that influence the content of a major bioactive component, saikosaponin. To identify genes associated with content and total yield of saikosaponin, it is of key importance to select stable references in gene expression analyses using quantitative real-time polymerase chain reaction (qRT-PCR). In this study, 18 candidate reference genes were selected and evaluated through their expression stability during the lateral root development in tissue samples from B. chinense DC., B. falcatum L. and B. scorzonerifolium Willd. The GeNorm, NormFinder and Bestkeeper methods were used for selecting stably expressed internal controls in the three Bupleurum species. These results revealed that, among these 18 candidate reference genes, ADF7 showed the best performance in all the experimental systems. ADF5 and ADF1b could also be proposed as suitable reference genes for gene expression studies. This study supplied more candidate reference genes to monitor the content and yield of saikosaponin during lateral roots growth in the Bupleurum genus.

Transcriptome-wide identification of optimal reference genes for expression analysis of Pyropia yezoensis responses to abiotic stress

BackgroundPyropia yezoensis, a marine red alga, is an ideal research model for studying the mechanisms of abiotic stress tolerance in intertidal seaweed. Real-time quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method to analyze gene expression levels. To accurately quantify gene expression, selection and validation of stable reference genes is required.ResultsWe used transcriptome profiling data from different abiotic stress treatments to identify six genes with relatively stable expression levels: MAP, ATPase, CGS1, PPK, DPE2, and FHP. These six genes and three conventional reference genes, UBC, EF1-α, and eif4A, were chosen as candidates for optimal reference gene selection. Five common statistical approaches (geNorm, ΔCt method, NormFinder, BestKeeper, and ReFinder) were used to identify the stability of each reference gene. Our results show that: MAP, UBC, and FHP are stably expressed in all analyzed conditions; CGS1 and UBC are stably expressed under conditions of dehydration stress; and MAP, UBC, and CGS1 are stably expressed under conditions of temperature stress.ConclusionWe have identified appropriate reference genes for RT-qPCR in P. yezoensis under different abiotic stress conditions which will facilitate studies of gene expression under these conditions.