Endogenous Reference Research Articles

The quantitative determination method of covalently closed circular DNA HBV in puncture biopsy specimens of the liver.

To analyze the method HBV covalent-closed circular DNA quantitative determination in liver puncture biopsies and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Republic of Uzbekistan. For quantitative analysis of HBV covalently closed circular DNA in a biopsy material a method was developed based on real-time PCR using TaqMan probes for the target fragment and for the endogenous reference gene, based on the detecting ccc HBV DNA method of Pollicino T. et al. When quantifying ccc DNA HBV in liver tissue of 18 moderately HBV activity with HBV DNA PCR positive results patients and 16 inactive HBsAg carriers, the ccc DNA HBV content was significantly different between groups (p<0.034) and in terms 1 copy of the β-globin gene among moderate activity HBV patients amounted to 1.71±1.32 copies/cell, and for inactive HBsAg carriers 0.15±0.14 copies/cell. In the group of patients with severe liver fibrosis and cirrhosis, the amount of ccc DNA HBV in liver tissue in patients with HBV averaged 2.5±0.4 copies/cell, in patients with HBV + D on average 0.7±0.25 copies/cell, in patients with HCV + HBV co-infection 0.45±0.07 copies/cell, in patients with a preliminary diagnosis of chronic hepatitis C hepatitis, on average 0.12±0.04 copies/cell, in patients with cryptogenic hepatitis 0.2± 0.05 copies/cell. A significant difference was shown between the group of patients with chronic hepatitis B with marked fibrosis and cirrhosis of the liver with other patients groups, except for the group of 18 moderate activity chronic hepatitis B patients. The values of Student's t-test when compared with other groups were respectively: for patients with a HCV preliminary diagnosis t=5,92 p<0,05 f = 19, patients with cryptogenic hepatitis t=5,71 p<0,05 f = 18, with «inactive HBsAg carriage» t=5,55 p<0,05 f = 29, with HCV + HBV co-infection t=5,05 p<0,05 f = 15 and HBV + D co-infection t=3,82 p<0,05 f = 17. The covalently closed circular DNA HBV quantitative assessment method in liver puncture biopsies allows identifying HBsAgnegative chronic viral hepatitis B forms and also reflects the virus replication activity, which, in turn, makes it possible to assume further disease progression and evaluate the antiviral therapy effectiveness.

Human Circulating miRNAs Real-time qRT-PCR-based Analysis: An Overview of Endogenous Reference Genes Used for Data Normalization.

miRNAs are small non-coding RNAs of about 18–25 nucleotides that negatively regulate gene expression at the post-transcriptional level. It was reported that a deregulation of their expression patterns correlates to the onset and progression of various diseases. Recently, these molecules have been identified in a great plethora of biological fluids, and have also been proposed as potential diagnostic and prognostic biomarkers. Actually, real time quantitative polymerase chain reaction is the most widely used approach for circulating miRNAs (c-miRNAs) expression profiling. Nevertheless, the debate on the choice of the most suitable endogenous reference genes for c-miRNAs expression levels normalization is still open. In this regard, numerous research groups are focusing their efforts upon identifying specific, highly stable, endogenous c-mRNAs. The aim of this review is to provide an overview on the reference genes currently used in the study of various pathologies, offering to researchers the opportunity to select the appropriate molecules for c-miRNA levels normalization, when their choosing is based upon literature data.

Exogenous Endowment—Endogenous Reference Point

Abstract We develop a model in the presence of categories that creates a link between the agent’s exogenous endowment and an endogenous reference point. The reference point is the best feasible alternative in the endowment’s category. This reference generates a constraint set from which the final choice is made according to utility maximisation. The model predicts category bias, which generalises the status quo bias by attracting the agent to her endowment’s category but not necessarily to the endowment itself. We show that it accommodates recent experimental findings regarding the presence and absence of status quo bias in the realm of uncertainty. In a stylised financial setup, we show that it may lead to a risk premium even with risk-neutral agents.

Serous IFNA3 predicts unfavorable prognosis in lung cancer via abnormal activation of AKT signaling.

This study addresses the demand through datamining The Cancer Genome Atlas (TCGA) database and elucidates mechanistic involvements of interferon alpha 8 (IFNA8) in lung cancer. The overall survival and disease-free survival of lung cancer patients in respect to IFNA8 expression level were analyzed. IFNA8 expression levels in both serum and tumor tissue were determined by real-time polymerase chain reaction. The diagnostic value of serous IFNA8 in lung cancer was assessed by receiver operating characteristic (ROC) curve analysis. Cell viability and proliferation were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Cell Counting Kit-8 assays. in vivo pro-tumor effect of IFNA8 was evaluated using xenograft tumor model. The metastasis-prone behaviors were determined by Transwell chamber assay and tail vein-injection in mice. Protein levels of p-AKT, total AKT, and endogenous reference actin were analyzed by western blot. We uncovered high IFNA8 associated with unfavorable overall survival and disease-free survival in lung cancer patients from TCGA. We further characterized the aberrant over-expression of IFNA8 in both peripheral blood and solid tumor from our clinical patient panel, and ROC analysis suggested its potential diagnostic value. Ectopic over-expression of IFNA8 promoted viability and proliferation in both A549 and H1299 cells in vitro and accelerated xenograft tumor growth in vivo. Furthermore, IFNA8 facilitated migration, invasion, and metastasis of A549 cells in vivo. Mechanistically, we disclosed the over-activation of AKT signaling in IFNA8-proficient A549 cells, inhibition of which completely abolished the pro-tumor effects of IFNA8. We have identified IFNA8 as a novel biomarker for either diagnostic or prognostic purpose in lung cancer, which is mechanistically associated with abnormal activation of AKT signaling.

Self serving reference points in double auctions

AbstractWe present a novel approach for modelling self serving bias by way of reference dependence. Bias is modelled as a systematic individual level deviation of the endogenous reference point from the expected price of a good in a two person k‐double auction. We find that bias decreases the efficiency of the model, and that efficiency is decreasing in both the level of bias and reference dependence.

Dynamic pricing under competition with data-driven price anticipations and endogenous reference price effects

Online markets have become highly dynamic and competitive. Many sellers use automated data-driven strategies to estimate demand and to update prices frequently. Further, notification services offered by marketplaces allow to continuously track markets and to react to competitors’ price adjustments instantaneously. To derive successful automated repricing strategies is challenging as competitors’ strategies are typically not known. In this paper, we analyze automated repricing strategies with data-driven price anticipations under duopoly competition. In addition, we account for reference price effects in demand, which are affected by the price adjustments of both competitors. We show how to derive optimized self-adaptive pricing strategies that anticipate price reactions of the competitor and take the evolution of the reference price into account. We verify that the results of our adaptive learning strategy tend to optimal solutions, which can be derived for scenarios with full information. Finally, we analyze the case in which our learning strategy is played against itself. We find that our self-adaptive strategies can be used to approximate equilibria in mixed strategies.

The reference point in dynamic Prospect-based User Equilibrium: a simulation study

ABSTRACT In this paper, we revisit the concept of Prospect-based User Equilibrium in a dynamic context. We consider a mesoscopic Lighthill-Whitham-Richards (LWR) traffic model to determine time-dependent route costs. We propose a solution algorithm to determine the network equilibria. Monte Carlo simulations are used to account for the travel time distributions. We analyze the dynamic Prospect-based User Equilibrium compared to the benchmarks Deterministic and Stochastic User Equilibrium, on a synthetic Manhattan network. We set four endogenous reference points. We show that the setting of the reference point plays a very important role in the route flow patterns and on the network performance at an aggregated level, i.e. in terms of vehicles mean speed as well as internal and outflow capacities. Our results also enhance that the Prospect-based User Equilibrium is more sensitive to a change in the reference point than in the calibration of the users’ risk-aversion and risk-seeking parameters.

Analysis of RNA in the estimation of post-mortem interval: a review of current evidence.

Post-mortem interval (PMI) determination is one of the most important issues in forensic sciences. In the past, forensic scientists provided different approaches (physical, chemical, and entomological) for the estimation of PMI without success.However, advances in molecular biology over the last two decades have allowed us to assess the time-dependent degradation of biological markers (e.g., proteins, DNA, and RNA). Thus, the aim of the manuscript is to provide a review of the recent progress in the estimation of PMI using molecular biology methods, mainly focusing on the potential usefulness of RNA markers. To this end, 29 studies have been systematically reviewed, each one chosen according to specific inclusion/exclusion criteria. The selected studies evaluated the contribution of endogenous reference genes in different biological samples in order to determine the PMI based on post-mortem RNA degradation as a function of other influencing factors such as time, cause of death, and environmental conditions.

Validation of endogenous reference genes in rat cerebral cortex for RT-qPCR analyses in developmental toxicity studies

Relative gene expression data obtained from quantitative RT-PCR (RT-qPCR) experiments are dependent on appropriate normalization to represent true values. It is common to use constitutively expressed endogenous reference genes (RGs) for normalization, but for this strategy to be valid the RGs must be stably expressed across all the tested samples. Here, we have tested 10 common RGs for their expression stability in cerebral cortex from young rats after in utero exposure to thyroid hormone (TH) disrupting compounds. We found that all 10 RGs were stable according to the three algorithms geNorm, NormFinder and BestKeeper. The downstream target gene Pvalb was significantly downregulated in brains from young rats after in utero exposure to propylthiouracil (PTU), a medicinal drug inhibiting TH synthesis. Similar results were obtained regardless of which of the 10 RGs was used for normalization. Another potential gene affected by developmental TH disruption, Dio2, was either not affected, or significantly upregulated about 1.4-fold, depending on which RG was used for normalization. This highlights the importance of carefully selecting correct RGs for normalization and to take into account the sensitivity of the RT-qPCR method when reporting on changes to gene expression that are less than 1.5-fold. For future studies examining relative gene expression in rat cerebral cortex under toxicological conditions, we recommend using a combination of either Rps18/Rpl13a or Rps18/Ubc for normalization, but also continuously monitor any potential regulation of the RGs themselves following alterations to study protocols.

Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

Selection of suitable reference genes for core clock gene expression analysis by real-time qPCR in rat ovary granulosa cells.

Selection of a suitable endogenous reference gene is essential for investigating expression of clock genes Bmal1, Clock, Pers, Crys, Rev-erbα/β, and RORα/β/γ involved in the circadian system. In this study, we treated rat ovary granulosa cells with dexamethasone to synchronize circadian oscillation in vitro and determined expression levels of Bmal1 and Per2 and six candidate reference genes (Actb, Beta actin; B2m, Beta-2-microglobulin; Ppia, Cyclophilin A; Gapdh, Glyceraldehyde-3-phosphate dehydrogenase; Hprt, Hypoxanthine guanine phosphoribosyl transferase and Tbp, TATA-box-binding protein) using quantitative real-time PCR. We then employed three software programs, GeNorm, NormFinder, and BestKeeper, to analyze the expression data for the selection of the best reference gene. According to GeNorm, Tbp and B2m were assessed as the most stable reference genes; Tbp and Hprt were best by NormFinder and BestKeeper, respectively. Thus, we recommend Tbp as the most suitable reference gene for studying clock genes expression in rat ovary granulosa cells in vitro.

Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations.

BackgroundQuantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantifying mRNA abundance. With relative expression analysis, however, reliable data output is dependent on stably expressed reference genes across the samples being studied. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes.PurposeThis study was aimed to investigate the optimal reference genes for PCR in ARM rat models.MethodsWe selected 15 commonly used reference genes (Rps18, Actb, B2m, Gapdh, Ppia, Hprt1, Pgk1, Ywhaz, Tbp, Ubc, Rps16, Rpl13a, Rplp1, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. The expression stability and variability of these transcripts were subsequently evaluated using four methods (geNorm, NormFinder, comparative ΔCt, and BestKeeper).ResultsThe abundance of the candidate reference genes was qualified by RT-qPCR and the cycle threshold (Ct) values ranged between 14.07 (Rplp1) and 21.89 (Sdha). In the overall candidate genes, different variations existed across the different algorithms. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps18, Sdha, and Hmbs.ConclusionsThe most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps18 in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses.

Identification of suitable reference microRNA for qPCR analysis in pediatric inflammatory bowel disease.

Pediatric inflammatory bowel disease (IBD) accounts for 10-15% of IBD and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA, miR), small noncoding RNA molecules that modulate gene expression, have been the target of research in IBD diagnosis, surveillance, and therapy. Proper selection of reference genes, which are a prerequisite for accurate measurement of miRNA expression, is currently lacking. We hypothesize that appropriate normalization requires unique reference genes for different tissue and disease types. Through the study of 28 pediatric intestinal samples, we sought to create a protocol for selection of suitable endogenous reference genes. Candidate reference genes (miR-16, 193a, 27a, 103a, 191) were analyzed by RT-quantitative (q)PCR. Criteria used for designation of suitable reference genes were as follows: 1) ubiquitous: present in all tissue samples with quantification cycle value 15-35; 2) uniform expression: no differential expression between control and disease samples (P > 0.05); 3) stability: stability value <0.5 by NormFinder. Our results suggest the use of miR-27a/191 for Crohn's disease small bowel, none of the five candidate genes for Crohn's disease colon, and miR-16/27a for ulcerative colitis. Additionally, target miR-874 had differential expression when normalized with different reference genes. Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative.

Identification of potential diagnostic biomarkers for pneumonia caused by adenovirus infection in children by screening serum exosomal microRNAs.

Human adenovirus (HAdV) infection causes serious pneumonia in children, leading to significant morbidity and mortality rates. However, diagnostic biomarkers for HAdV-associated pneumonia are unavailable. Serum microRNAs (miRNAs/miRs) have been recently reported as diagnostic biomarkers for several diseases. The present study performed microRNA sequencing to identify potential biomarkers among serum exosomal miRNAs, with the aim of identifying candidate biomarkers for the diagnosis of pneumonia in adenovirus-infected children. To validate the biomarker candidates, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the relative expression levels of miRNAs. As there is no endogenous reference RNA for serum miRNAs, pairwise analysis of RT-qPCR was used in the present study to narrow down the number of biomarker candidates among all the serum exosomal miRNAs to a set of four miRNAs. As a result, the identified miRNAs (namely, miR-450a-5p-miR-103a-3p and miR-103b-5p-miR-98-5p) from 59 samples were considered as potential diagnostic biomarkers in adenovirus-infected children. The results indicated that this four miRNA set could distinguish adenovirus-infected patients from healthy controls. In conclusion, the four exosomal miRNAs identified in the present study could be considered as candidate diagnostic biomarkers for pneumonia in adenovirus-infected children.

Isotope ratio mass spectrometry in antidoping analysis: The use of endogenous reference compounds.

Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13 C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers. Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)-IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11-keto-etiocholanolone and 11β-hydroxyandrosterone. The obtained 13 C delta values were statistically analyzed to evaluate their inter- and intra-individual distribution. The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra-individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway. For the selected ERCs of non-sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13 C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts.

Plasma Level of miR-5193 As a Novel Biomarker for Diagnosis of HBV-Related Hepatocellular Carcinoma

Background: Chronic infection with hepatitis B virus (HBV) is related to more than 50% of all cases of hepatocellular carcinoma (HCC). The HBx protein of the virus plays an important role in carcinogenesis through different mechanisms, including interaction with the Notch1 signaling pathway. Several microRNAs have also been shown to play essential roles in the carcinogenesis of HCC, and the molecules can be considered the novel biomarkers for the diagnosis of different types of cancer. Methods: In the present study, the expression levels of four bioinformatically predicted microRNAs, including miR-214, miR-6510, miR-5193, and miR-34a, were compared in individuals with HBV-associated HCC and controls in order to assess the diagnostic utility of these microRNAs as noninvasive biomarkers. Seventy three plasma samples were subjected to RNA extraction, and the microRNA expression profiles were assessed by RT-qPCR. The expression of miR-103 was used as the endogenous reference for the normalization of quantitative data. Results: The plasma expression of all miRNAs was significantly lower in the HCC group, but the downregulation was most marked for the newly described molecule, miR-5193 (-18.86 folds and -5.71 folds compared to healthy individuals and chronic HBV patients). Another novel microRNA, miR-6510, was downregulated by -4.23 folds (P = 0.001). The results of the ROC curve analysis indicated that the differential expression of miR-5193 could distinguish HCC patients with high sensitivity and specificity. Conclusions: The study microRNAs may have a role in HCC development and progression. In addition, miR-5193 can be potentially used as a non-invasive biomarker for the detection of HBV-induced HCC.

Development of a mass spectrometry based detection method for the mitochondrion-derived peptide MOTS-c in plasma samples for doping control purposes.

The mitochondrial open reading frame of 12S rRNA type-c (MOTS-c) peptide was recently discovered and described to control metabolic homeostasis through AMPK activation along with AICAR accumulation. Consequently, it appears advisable to monitor the potential use of synthetic MOTS-c in sports, and a detection method suitable for sports drug testing purposes is necessary. For the detection of MOTS-c in doping control plasma samples, a test method employing liquid chromatography and mass spectrometry (LC/MS) was developed. Following optimization, the assay was comprehensively validated and additional parameters such as the (long-term) stability and in vitro metabolism of the peptide were evaluated. In order to determine endogenous MOTS-c reference limits, the results generated by LC/MS-based detection were compared with those obtained with a commercially available enzyme-linked immunosorbent assay (ELISA). The LC/MS-based test method was fully validated for quantitative results interpretation according to the World Anti-Doping Agency's International Standard for Laboratories (WADA's ISL). It was found to be specific and sensitive, enabling a lower limit of detection (LLOD) for hMOTS-c in plasma at 100 pg/mL. Following optimization, animal MOTS-c analogues and four metabolites as well as two oxidation products were implemented. However, endogenous levels of a reference population of 20 healthy subjects studied by ELISA experiments (45.9-218.5 ng/mL) could not be confirmed by LC/MS. A mass spectrometric detection assay for MOTS-c in human plasma samples was developed and successfully validated according to WADA's ISL, providing an additional tool for future doping control purposes. Besides MOTS-c, the assay also includes four in vitro derived metabolites and two oxidation products, which might further improve the traceability of the drug. The analytical approach was compared with a commercially available ELISA, and considerable differences in measured MOTS-c levels were observed.

Expectations-Based Reference-Dependence and Choice Under Risk

Abstract This article characterises the behavioural content of a model of choice under risk with reference-dependent preferences and endogenous expectations-based reference points based on the preferred personal equilibrium model of Kőszegi and Rabin (2006). The combination of reference-dependent preferences and endogenous reference points leads to violations of the Independence Axiom and can also lead to violations of the Weak Axiom of Revealed Preference. An axiomatic characterisation shows that the model places testable restrictions on choice under risk.

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation.

Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.

Selection of endogenous reference genes for qRT-PCR analysis in Camelina sativa and identification of FLOWERING LOCUS C allele-specific markers to differentiate summer- and winter-biotypes

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis greatly relies on transcript normalization using stably expressed reference genes. This study identifies reference genes for qRT-PCR analysis in different organs and vernalized tissues of camelina (Camelina sativa). Nineteen stably expressed candidate reference genes from 22,157 genes were identified by RNAseq analysis of pre- and post-vernalized tissues of a summer- (CO46) and winter- (Joelle) biotype. Two additional candidate reference genes were also selected from orthologs of arabidopsis encoding ACTIN2 (ACT2) and NOT INDUCED BY SCLEROTINIA INFECTION (NIS). We also evaluated the transcript levels of three camelina plant genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), FLOWERING LOCUS C (FLC), and MADS AFFECTING FLOWERING 2 (MAF2), which are known to be differentially-regulated in response to cold temperatures. The stability of transcript abundance was ranked using NormFinder, geNorm, BestKeeper, and Comparative ΔCT software. EXOCYST COMPLEX COMPONENT SEC3A (SEC3A), UBIQUINONE OXIDOREDUCTASE (UbOxRed), and RING-U-BOX domains-containing protein (RUB) were the most suitable reference genes for normalization of gene expression in camelina. In this study, SEC3A was used to normalize qRT-PCR expression data obtained using two sets of allele-specific FLC markers to differentiate summer- and winter-biotypes across 30 accessions of camelina.