Normalization Of qRT-PCR Data Research Articles

Proposal of reference genes for tomato fruit ripening qRT-PCR data normalization.

Proposal of reference genes for tomato fruit ripening qRT-PCR data normalization.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Mythimna loreyi (Lepidoptera: Noctuidae).

Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA-box, GAPDH, AK, Actin, EF, α-tubulin, SOD, 18S rRNA, and FTZ-F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ-F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.

Reference gene selection for quantitative real-time PCR analysis of Hymenopellis radicata under abiotic stress

Hymenopellis radicata (H. radicata) is an edible fungus rich in protein and mineral elements, with high edible and medical value. And reference genes suitable for normalization of qRT-PCR data from this species have not been investigated. In this study, therefore, we selected 11 housekeeping genes common in biology. The expression levels of these housekeeping genes were measured in three different tissues and six different abiotic stress treatments in mycelium. They were evaluated for expression stability using online tools. The results showed that gene ACT could be stable expressed in all samples. The expressions of genes TUB and UBQ10 are the most stable under heat stress, ACT and EF are the most stable genes under salt stress, ACT and TUB are the most stable genes under oxidation stress, RPL6 and EF are the most stable genes under pH condition, ACT and RPB2 are the most stable genes under cadmium stress, and RPB2 and UBC are the most stable genes under drought condition. ACT and PP2A are the most stable genes at different tissue sites. This study is of great help to explore the gene expression pattern of H. radicata, and also provides reference for internal reference gene screening under other conditions.

Screening of Reference Genes under Biotic Stress and Hormone Treatment of Mung Bean (Vigna radiata) by Quantitative Real-Time PCR.

Mung bean (Vigna radiata) production has been greatly threatened by numerous diseases. Infection with these pathogens causes extensive changes in gene expression and the activation of hormone signal transduction. Quantitative real-time PCR (qRT-PCR) is the most common technique used for gene expression validation. Screening proper reference genes for mung bean under pathogen infection and hormone treatment is a prerequisite for ensuring the accuracy of qRT-PCR data in mung bean disease-resistance research. In this study, six candidate reference genes (Cons4, ACT, TUA, TUB, GAPDH, and EF1α) were selected to evaluate the expression stability under four soil-borne disease pathogens (Pythium myriotylum, Pythium aphanidermatum, Fusarium oxysporum, and Rhizoctonia solani) and five hormone treatments (SA, MeJA, ETH, ABA, and GA3). In the samples from different treatments, the Ct value distribution of the six candidate reference genes was different. Under the condition of hormone treatment, the Ct value ranged from a minimum of 17.87 for EF1α to a maximum of 29.63 for GAPDH. Under the condition of pathogen infection, the Ct value ranged from a minimum of 19.43 for EF1α to a maximum of 31.82 for GAPDH. After primer specificity analysis, it was found that GAPDH was not specific, so the five reference genes Cons4, ACT, TUA, TUB, and EF1α were used in subsequent experiments. The software products GeNorm, NormFinder, BestKeeper and RefFinder were used for qRT-PCR data analysis. In general, the best candidates reference genes were: TUA for SA, ABA, GA3, and Pythium myriotylum treatment; TUB for ETH treatment; ACT for MeJA and Fusarium oxysporum treatment; and EF1α for Pythium aphanidermatum and Rhizoctonia solani treatment. The most stably expressed genes in all samples were TUA, while Cons4 was the least stable reference gene. Finally, the reliability of the reference gene was further validated by analysis of the expression profiles of four mung bean genes (Vradi0146s00260, Vradi0158s00480, Vradi07g23860, and Vradi11g03350) selected from transcriptome data. Our results provide more accurate information for the normalization of qRT-PCR data in mung bean response to pathogen interaction.

Selection of Reference Genes for Normalization of qRT‒PCR Analysis in the Soybean Aphid Aphis glycines Matsumura (Hemiptera: Aphididae).

The soybean aphid Aphis glycines Matsumura is a predominant insect pest in Asia and North America and causes great losses to soybean. The release of genome data for A. glycines will facilitate gene function research in the future. However, suitable reference genes for A. glycines under various experimental conditions are scarce. To search for appropriate reference genes for A. glycines, nine candidate reference genes, including Act, α-Tub, β-Tub, RPS12, RPS18, RPL5, RPL27, EF1α, and Fer, were tested under six experimental conditions to evaluate their suitability for use in the normalization of qRT‒PCR data. Results showed that EF1α and RPS12 were optimal for the developmental stages of A. glycines, RPS18 and RPS12 were appropriate for wing dimorphism, β-Tub and RPS18 were suitable for different tissues and RPL5, and α-Tub could be used for normalization at different temperatures. β-Tub and EF1α could be proposed as reference genes for insecticide treatment, and RPL5 and RPS12 were found to be the most stable reference genes in different photoperiods. The results provide appropriate reference genes for analyzing gene expression in A. glycines and contribute to future research on the molecular physiology and biochemistry of A. glycines.

Reference gene selection for transcriptional profiling by RT-qPCR in the 28-spotted larger potato ladybird

• The stabilities of nine house-keeping genes are studied under four conditions. • RPL6 and RPL13 are for different developmental stages. • RPS18 and EF1α are for different tissues. • RPS18 and RPL13 are for four different experimental conditions. • Our findings establish an accurate normalization of qRT-PCR data. Henosepilachna vigintioctomaculata is one of the most serious defoliates attacking potatoes. However, studies on functional genes have greatly been limited due to the insufficiency of effective and stable endogenous references to normalize RT-qPCR data. In this report, nine housekeeping genes ( RPL4 , RPL6 , RPL13 , RPL32 , RPS18 , ACT , EF1α , GAPDH and α-TUB ) involved in different biological processes were selected. Their expression levels under diverse experimental conditions including developmental stages, tissues, temperatures and host plants were determined using RT-qPCR technology. The tested candidate genes were comprehensively ranked based on five alternative stability analysis methods (Ct value, geNorm, NormFinder, BestKeeper and ReFinder). The results revealed that the optimal internal reference genes varied under different experimental conditions. Any gene pair among the five candidates ( RPL4, RPL13, RPL32, RPS18 and EF1α ) was a suitable reference gene set under different temperatures and on different host plants. A combination of RPL6 and RPL13 was recommended as the best reference gene set across different developmental stages. A pair of RPS18 and EF1α was ranked as the optimal reference gene combination within different tissues. The most suitable reference genes were RPS18 and RPL13 under four different experimental conditions. Our findings not only establish an accurate and reliable normalization of RT-qPCR data, but also lay a solid foundation for further functional gene researches in H. vigintioctomaculata .

Identification of Suitable Reference Genes for qRT-PCR Normalization in Kiwifruit

Reference genes are used for the correction of qRT-PCR data, and it is necessary to investigate the optimum reference gene under certain conditions. The expression levels of seven traditional reference genes ACT1, ACT2, GAPDH, 18S rRNA, UBQ, TUB and CYP were analyzed using qRT-PCR in different varieties, tissues, developmental stages and hormone (or pollen polysaccharide) treatments in kiwifruit. Gene expression stability was assessed with the help of three common software (geNorm, NormFinder, BestKeeper), and the minimum number of reference genes necessary for normalization was also determined. GAPDH, ACT1 and ACT2 were selected as reference genes for different genotypes of kiwifruit. GAPDH and UBQ were the best combinations of reference genes for root, stem, leaf, flower and fruit. GAPDH and ACT1 could be the preferred reference genes for normalization of qRT-PCR data during fruit development. The pairing of ACT1 and UBQ constituted the optimal combination of reference genes in kiwifruit treated with different hormones (or pollen polysaccharide). This study provides a new and reliable option for the use of reference genes in the analysis of gene expression patterns of interest in kiwifruit.

Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp.

There has been significant interest in researching the pharmaceutical applications of Industrial hemp since its legalization three years ago. The crop is mostly dioecious and known for its production of phytocannabinoids, flavonoids, and terpenes. Although many scientific reports have showed gene expression analysis of hemp through OMICs approaches, unreliable reference genes for normalization of qRT-PCR data make it difficult to validate the OMICs data. Four software packages: geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stresses. EF-1α ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal stress and hormonal stimuli. The expression patterns of two cannabinoid pathway genes, AAE1 and CBDAS, were used to validate the reliability of the selected reference genes. This work provides useful information for gene expression characterization in hemp and future research in the synthesis, transport, and accumulation of secondary metabolites.

Putative Internal Control Genes in Bovine Milk Small Extracellular Vesicles Suitable for Normalization in Quantitative Real Time-Polymerase Chain Reaction.

Bovine milk small extracellular vesicles (sEVs) contain many biologically important molecules, including mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for quantifying mRNA in tissues and cells. However, the use, selection, and stability of suitable putative internal control genes in bovine milk sEVs for normalization in qRT-PCR have not yet been identified. Thus, the aim of the present study was to determine suitable putative internal control genes in milk sEVs for the normalization of qRT-PCR data. Milk sEVs were isolated from six healthy Holstein-Friesian cattle, followed by RNA extraction and cDNA synthesis. In total, 17 mRNAs were selected for investigation and quantification using qRT-PCR; they were further evaluated using geNorm, NormFinder, BestKeeper, and ∆CT algorithms to identify those that were highly stable putative internal control genes in milk sEVs. The final ranking of suitable putative internal control genes was determined using RefFinder. The mRNAs from TUB, ACTB, DGKZ, ETFDH, YWHAZ, STATH, DCAF11, and EGFLAM were detected in milk sEVs from six cattle by qRT-PCR. RefFinder demonstrated that TUB, ETFDH, and ACTB were highly stable in milk sEVs, and thus suitable for normalization of qRT-PCR data. The present study suggests that the use of these genes as putative internal control genes may further enhance the robustness of qRT-PCR in bovine milk sEVs. Since these putative internal control genes apply to healthy bovines, it would be helpful to include that the genes were stable in sEVs under “normal or healthy conditions”.

Selection of Suitable Reference Genes for qRT-PCR Gene Expression Studies in Rice.

With a widely established use of quantitative real-time PCR (qRT-PCR) for gene expression analysis, reliable and stable expression of reference genes is often discussed. Suitable reference genes should show less variation of expression across the target samples and allow for error minimization by normalization of qRT-PCR data. Therefore, selection of reliable reference genes is essential for accurate results and to support the conclusions drawn on expression levels of genes under study. In this chapter, we describe the workflow for selection and evaluation of reference genes in rice, including identification of candidate genes by using Genevestigator® and evaluation of expression stability using various algorithms. The ranking of the genes guides qRT-PCR performance and data analysis. This protocol used rice as an example but is not limited to rice, and could be applied to other species as well.

Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica.

Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is the most sensitive approach to produce accurate relative gene expression that has yet to be demonstrated for allergenic tropomyosin in mud crab species. This study was conducted to identify the suitable reference gene and tropomyosin expression in different body parts of local mud crabs, Scylla olivacea, Scylla paramamosain and Scylla tranquebarica. Myosin, 18S rRNA, GADPH and EF1α were selected as candidate reference genes and their expression was measured in the abdomen, walking leg and cheliped tissues of local Scylla spp. The expression stability was analyzed using the comparative delta-Ct method, BestKeeper, NormFinder and geNorm then comprehensively ranked by RefFinder algorithm. Findings showed that EF1α was the most suitable reference gene across three mud crab species. Meanwhile, the abdomen, walking leg and cheliped selected their own suitable reference gene either Myosin, 18S rRNA, EF1α or GADPH. Overall, tropomyosin was the highest in S. tranquebarica, whereas the least was in S. paramamosain. Interestingly, tropomyosin was the highest in the abdomen of all mud crab species. This is the first analysis on reference genes selection for qRT-PCR data normalization of tropomyosin expression in mud crab. These results will provide more accurate findings for further gene expression and allergen analysis in Scylla spp.

Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xanthomonas\xa0oryzae\xa0pv. oryzae--infection and melatonin supplementation

Exogenous melatonin (MT) was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv. oryzae (Xoo)-caused bacterial blight (BB). However, the accurate comparison of the expression levels across samples was a challenging task. In this work, the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR (qRT-PCR), and algorithms geNorm, NormFinder and BestKeeper. Our results indicated that most reference genes remained stable in Xoo-infected rice plants, while a number of reference genes were affected by MT supplementation. Among all studied genes, the transcript levels of 18S(18S ribosomal RNA) and UBC (Ubiquitin-conjugating enzyme E2) remained unaltered by Xoo infection, while UBC and UBQ5(Ubiquitin 5) were the most stable genes when examining simultaneous Xoo-infection and MT supplementation, demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.

Selection and Validation of Reference Genes for the qRT-PCR Assays of Populus ussuriensis Gene Expression under Abiotic Stresses and Related ABA Treatment

Populus ussuriensis Kom. is one of the most important tree species for forest renewal in the eastern mountainous areas of Northeast China due to its fast growth, high yield, and significant commercial and ecological value. The selection of optimal reference genes for the normalization of qRT-PCR data is essential for the analysis of relative gene expression. In this study, fourteen genes were selected and assessed for their expression stability during abiotic stress (drought, high salinity, and cold stress) and after the treatment with the drought-related hormone ABA. Three algorithms were used, geNorm, NormFinder, and BestKeeper, and a comprehensive ranking of candidate reference genes was produced based on their output. The most appropriate reference genes were UBQ10 and RPL24 for drought and ABA treatment, UBQ10 and TUB3 for cold stress, and UBQ10 and 60S rRNA for high salinity. Overall, UBQ10 was the most stable reference gene for use as an internal control, whereas PP2A was the least stable. The expression of two target genes (P5CS2 and GI) was used to further verify that the selected reference genes were suitable for gene expression normalization. This work comprehensively assesses the stability of reference genes in Populus ussuriensis and identifies suitable reference genes for normalization during qRT-PCR analysis.

Identification and validation of superior housekeeping gene(s) for qRT-PCR data normalization in Agave sisalana (a CAM-plant) under abiotic stresses.

The adaptive mechanisms in Agave species enable them to survive and exhibit remarkable tolerance to abiotic stresses. Quantitative real-time PCR is a highly reliable approach for validation of targeted differential gene expression. However, stable housekeeping gene(s) is prerequisite for accurate normalization of expression data by qRT-PCR. Till date, no systematic validation study for candidate housekeeping gene identification or evaluation has been carried-out in Agavespecies. A total of 17 candidate housekeeping genes were identified from the de novo assembled transcriptomic data of A. sisalana and rigorously analyzed for expression stability assessment under drought, heat, cold and NaCl stress. Different statistical algorithms like geNorm, BestKeeper, NormFinder, and RefFinder on expression data determined the superior housekeeping gene(s) for accurate normalization of the gene of interest (GOI). The comprehensive evaluation revealed the β-Tub 4, WIN-1 and CYC-A as the most stable, while EEF1α, GAPDH, and UBE2 were ranked as the least stable genes in pooled samples. Pairwise combination by geNorm showed that up to two housekeeping genes would be adequate to normalize the GOI expression data precisely. Validation of identified most and least stable housekeeping genes was carried-out by normalizing the expression data of AsHSP20 under abiotic stress conditions. Copy number of AsHSP20 gene supports the reliability of the genes used for normalization. This is the first report on the screening and validation of the housekeeping genes under abiotic stress condition in A. sisalana that would assist to understand the stress tolerance mechanisms by novel gene identification and accurate validation.

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus)

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18s), beta-actin (actb), elongation factor 1-alpha (ef1α), glyceraldehyde-3-phosphate-dehydrogenase (gapdh), cathepsin D (ctsd), and beta-2-microglobulin (b2m) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were ef1α, actb, and ctsd in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18s, ef1α, and ctsd in the hypothalamus; actb, ctsd, and 18s in the pituitary; actb, and ctsd in the ovary; gapdh and ctsd in the liver. Moreover, the expression profile of estrogen receptor α (erα) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development.

Reference Gene Selection for Quantitative Real-Time PCR Analyses of Acer palmatum under Abiotic Stress

Quantitative real-time reverse transcriptase PCR (qRT-PCR) technology has been extensively used to estimate gene expression levels, and the selection of appropriate reference genes for qRT-PCR analysis is critically important for obtaining authentic normalized data. Acer palmatum is an important colorful leaf ornamental tree species, and reference genes suitable for normalization of the qRT-PCR data obtained from this species have not been investigated. In this study, the expression stability of ten candidate reference genes, namely, Actin3, Actin6, Actin9, EF1α, PP2A, SAMDC, TIP41, TUBα, TUBβ and UBQ10, in two distinct tissues (leaves and roots) of A. palmatum under four different abiotic stress conditions (cold, heat, salt and drought) were investigated and assessed using three statistical methods (GeNorm, NormFinder and BestKeeper). The combinations of reference genes that showed stability in the different stressed samples differed. Specifically, Actin6, UBQ10 and Actin9 were the most stable reference genes in all the samples, and Actin3 and Actin6 wexre stably expressed in coldstressed leaves and roots. Actin3, Actin6 and UBQ10 were identified as an appropriate combination of reference genes for the analysis of heat-stressed leaves and roots, whereas the combination of Actin9, UBQ10 and Actin6 was deemed the most suitable for the analysis of salt-stressed leaves and roots. Similarly, Actin6 and UBQ10 exhibited stable expression in drought-stressed leaves and roots. Furthermore, the expression levels of CBF, Cu/Zn-SOD and HsfA1 were estimated to determine the reliability of the reference genes assessed in this study. This study revealed stable reference genes in A. palmatum that might be used for the normalization of qRT-PCR data obtained under various abiotic stresses.

Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice.

Accurate quantitative reverse transcription PCR (qRT-PCR) requires reliable reference genes whose expression does not vary in different tissues and developmental stages. However, few reliable reference genes are available for qRT-PCR in rice (Oryza sativa). Here, we established an effective strategy for identifying novel reference genes (NRGs) for reliable normalization of qRT-PCR data in various rice organs and developmental stages. We selected candidate NRGs using the Information Commons for Rice Database and confirmed their expression in Rice Expression Profile Database (RiceXPro) data. Genes with low variation (<2.5 cycle quantification) across tissues and developmental stages, and little fluctuation in expression in heatmaps from RiceXPro data were considered stable NRGs. To validate this strategy, we selected 11 candidate NRGs and calculated their expression stability in different spatio-temporal conditions using five programs, and compared these genes with five established reference genes (ERGs). Only one of the ERGs (UBQ5) was reliable and 10 of the candidate NRGs were more stable than the four remaining ERGs. Therefore, public transcriptomic databases are useful for identifying NRGs. We selected two NRGs, UFC1 (Homolog of UFM1-Conjugating Enzyme 1) and FhaB (Homolog of Adhesin FhaB) for qRT-PCR analysis in rice; their homologs might be suitable for other monocot plants.

Identification of stably expressed housekeeping miRNAs in endothelial cells and macrophages in an inflammatory setting

Reliable quantification of miRNA expression by qRT-PCR crucially depends on validated housekeepers for data normalization. Here we present thoroughly tested miRNAs eligible as references in immunological studies utilizing endothelial cells and macrophages, respectively. Endothelial cells (cell line: TIME) and macrophages (cell line: RAW264.7) were treated with various pro- and anti-inflammatory mediators (cytokines, LPS, unsaturated fatty acids) given as either single substances or in combination. Isolated RNA was screened for stably expressed miRNAs by next generation sequencing. Housekeeper candidates were thereafter validated by means of two independent quantification techniques: qRT-PCR for relative quantification and ddPCR for absolute quantification. Both methods consistently confirmed the suitability of let-7g-5p, let-7i-5p, miR-127-3p and miR-151a-5p in cytokine/fatty acid-treated TIME and miR-16-5p, miR-27b-3p, miR-103a-3p and miR-423-3p in LPS/fatty acid-treated RAW264.7, respectively as housekeeping miRNAs. With respect to abundancy and over all expression stability the miRNAs miR-151a-5p (cell line: TIME) as well as miR-27b-3p and miR-103a-3p (cell line: RAW264.7) can be particularly recommended for normalization of qRT-PCR data.

Validation of reference genes in human epicardial adipose tissue and left ventricular myocardium in heart failure

The characterization of tissue-specific gene expression in failing hearts requires the identification of appropriately validated reference genes. This study sought to validate which commonly used reference genes were the most suitable for qRT-PCR data normalization in epicardial adipose tissue and left ventricular myocardium in patients with end-stage heart failure. The mRNA expression of 10 candidate reference genes was analyzed by qRT-PCR. The stability of their expression was evaluated by our novel scoring system and compared with known algorithms used to identify stable reference genes (Normfinder and Bestkeeper). Using these three methods, we selected the Ribosomal protein L13A (RPL13A) as the best reference gene when studying either epicardial adipose tissue or left ventricular myocardium in failing hearts. In contrast, the β2-microglobulin (B2M) gene was identified as a more suitable reference gene in studies comparing gene expression in epicardial adipose tissue to left ventricular myocardium.

Selection of best reference genes for qRT-PCR analysis of human neural stem cells preconditioned with hypoxia or baicalein-enriched fraction extracted from Oroxylum indicum medicinal plant

Whilst the potential of neural stem cell (NSC)-based treatment is recognized worldwide and seems to offer a promising therapeutic option for stroke treatments, there is currently no full understanding regarding the effects of hypoxic and baicalein-enriched fraction (BEF) preconditioning approaches on the therapeutic potential of these cells for stroke. The potential of preconditioned NSC can be determined based on the expression of several key neuroprotective genes using qRT-PCR technique. However, prior to that, it is imperative and extremely important to carefully select reference gene(s) for accurate qRT-PCR data normalization to avoid error in data interpretation. This study aimed to evaluate the stability of ten candidate reference genes via comprehensive analysis using three algorithms software: geNorm, NormFinder and BestKeeper. Our results revealed that HPRT1 and RPL13A were the most reliable reference genes for BEF-preconditioned NSCs, but ironically, HPRT1 was ranked as the least stable reference gene for hypoxic-preconditioned NSCs. On the other hand, RPLP1 and RPL13A were selected as the most stably expressed pair of reference genes for hypoxic-preconditioned NSCs. In conclusion, this study has pointed out the importance of identifying valid reference genes and has presented the first significant validation on best reference genes recommended for qRT-PCR study involves NSC preconditioned with hypoxia or with BEF extracted from Oroxylum indicum medicinal plant.