Impacto del grado de cumplimiento de un protocolo ERAS en la recuperación funcional después de cirugía colorrectal / ERAS protocol compliance impact on functional recovery in colorectal surgery

INTRODUCCIÓN: El grado de cumplimiento de los protocolos de Enhanced Recovery After Surgery (ERAS) es una medida de calidad del proceso, que además se asocia a mejores resultados. El objetivo del presente estudio es analizar la relación existente entre el grado de cumplimiento del protocolo, el estrés quirúrgico y la recuperación funcional. Se plantea como objetivo secundario, la identificación de factores independientes asociados a la recuperación funcional. MÉTODOS: Estudio retrospectivo observacional unicéntrico de pacientes sometidos a cirugía colorrectal programada dentro de un programa ERAS entre enero de 2017 y junio de 2018. Se analizó el grado de cumplimiento del protocolo porcentual y su relación con el estrés quirúrgico (definido por los niveles plasmáticos de proteína C reactiva al tercer día), y la recuperación funcional (definida por el cumplimiento de los criterios de alta el quinto día postoperatorio o antes). Se llevó a cabo un análisis multivariante de factores independientes asociados a recuperación funcional. RESULTADOS: Se analizaron 313 pacientes. Por cada punto porcentual de cumplimiento adicional del protocolo disminuye 1,46 mg/dL la proteína C reactiva del tercer día y aumenta un 7% la probabilidad de cumplir criterios de alta (p < 00,1 ambos). Los factores asociados a recuperación funcional fueron ASA III-IV (OR 0,26; 0,14-0,48), puntuación CR-POSSUM quirúrgico (OR 0,68; 0,57-0,83), movilización precoz (OR 4,22; 1,43-12,4) y retirada precoz de sonda vesical (OR 3,35; 1,79-6,27), todos ellos p < 0,001. CONCLUSIÓN: El aumento del grado de cumplimiento del protocolo ERAS en cirugía colorrectal, disminuye el estrés quirúrgico y acelera la recuperación funcional

INTRODUCTION: Compliance to ERAS protocols is a process quality measure that is associated to better outcomes. The main objective of this study is to analyze the association between protocol compliance, surgical stress and functional recovery. The secondary objective is to identify independent factors associated to functional recovery. METHODS: A prospective observational single-centre study was performed. Patients who had scheduled colorectal surgery within an ERAS program from January 2017 to June 2018 were included. We analysed the relationship between protocol compliance percentage and surgical stress (defined by C reactive protein [CRP] blood levels on postoperative 3rd day), and functional recovery (defined by the proportion of patients who meet the discharge criteria on the 5th PO day or before). Multivariate analysis was performed to asses independent factor associated to functional recovery. RESULTS: 313 were included. For every additional percentage point of compliance to the protocol 3rd day C reactive protein plasmatic level decreases 1,46 mg/dL and increases 7% the probability to meet the discharge criteria (p < 0.001 both). Independent factors associated to functional recovery were ASA III-IV (OR 0.26; 0.14-0.48), surgical CR-POSSUM score (OR 0.68; 0.57-0.83), early mobilization (OR 4.22; 1.43-12.4) and removal of urinary catheter (OR 3.35; 1.79-6.27), p < 0,001 each of them. CONCLUSION: Better copliance to ERAS protocol in colorectal surgery decreases surgical stress and accelerates functional recovery

ERAS protocol compliance impact on functional recovery in colorectal surgery. / Impacto del grado de cumplimiento de un protocolo ERAS en la recuperación funcional después de cirugía colorrectal.

INTRODUCTION: Compliance to ERAS protocols is a process quality measure that is associated to better outcomes. The main objective of this study is to analyze the association between protocol compliance, surgical stress and functional recovery. The secondary objective is to identify independent factors associated to functional recovery. METHODS: A prospective observational single-centre study was performed. Patients who had scheduled colorectal surgery within an ERAS program from January 2017 to June 2018 were included. We analysed the relationship between protocol compliance percentage and surgical stress (defined by C reactive protein [CRP] blood levels on postoperative 3rd day), and functional recovery (defined by the proportion of patients who meet the discharge criteria on the 5th PO day or before). Multivariate analysis was performed to asses independent factor associated to functional recovery. RESULTS: 313 were included. For every additional percentage point of compliance to the protocol 3rd day C reactive protein plasmatic level decreases 1,46 mg/dL and increases 7% the probability to meet the discharge criteria (p < 0.001 both). Independent factors associated to functional recovery were ASA III-IV (OR 0.26; 0.14-0.48), surgical CR-POSSUM score (OR 0.68; 0.57-0.83), early mobilization (OR 4.22; 1.43-12.4) and removal of urinary catheter (OR 3.35; 1.79-6.27), p < 0,001 each of them. CONCLUSION: Better compliance to ERAS protocol in colorectal surgery decreases surgical stress and accelerates functional recovery.

First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.).

BACKGROUND: Zucchini fruit set can be limited due to unfavourable environmental conditions in off-seasons crops that caused ineffective pollination/fertilization. Parthenocarpy, the natural or artificial fruit development without fertilization, has been recognized as an important trait to avoid this problem, and is related to auxin signalling. Nevertheless, differences found in transcriptome analysis during early fruit development of zucchini suggest that other complementary pathways could regulate fruit formation in parthenocarpic cultivars of this species. The development of next-generation sequencing technologies (NGS) as RNA-sequencing (RNA-seq) opens a new horizon for mapping and quantifying transcriptome to understand the molecular basis of pathways that could regulate parthenocarpy in this species. The aim of the current study was to analyze fruit transcriptome of two cultivars of zucchini, a non-parthenocarpic cultivar and a parthenocarpic cultivar, in an attempt to identify key genes involved in parthenocarpy. RESULTS: RNA-seq analysis of six libraries (unpollinated, pollinated and auxin treated fruit in a non-parthenocarpic and parthenocarpic cultivar) was performed mapping to a new version of C. pepo transcriptome, with a mean of 92% success rate of mapping. In the non-parthenocarpic cultivar, 6479 and 2186 genes were differentially expressed (DEGs) in pollinated fruit and auxin treated fruit, respectively. In the parthenocarpic cultivar, 10,497 in pollinated fruit and 5718 in auxin treated fruit. A comparison between transcriptome of the unpollinated fruit for each cultivar has been performed determining that 6120 genes were differentially expressed. Annotation analysis of these DEGs revealed that cell cycle, regulation of transcription, carbohydrate metabolism and coordination between auxin, ethylene and gibberellin were enriched biological processes during pollinated and parthenocarpic fruit set. CONCLUSION: This analysis revealed the important role of hormones during fruit set, establishing the activating role of auxins and gibberellins against the inhibitory role of ethylene and different candidate genes that could be useful as markers for parthenocarpic selection in the current breeding programs of zucchini.

Multiclass classification for skin cancer profiling based on the integration of heterogeneous gene expression series.

Most of the research studies developed applying microarray technology to the characterization of different pathological states of any disease may fail in reaching statistically significant results. This is largely due to the small repertoire of analysed samples, and to the limitation in the number of states or pathologies usually addressed. Moreover, the influence of potential deviations on the gene expression quantification is usually disregarded. In spite of the continuous changes in omic sciences, reflected for instance in the emergence of new Next-Generation Sequencing-related technologies, the existing availability of a vast amount of gene expression microarray datasets should be properly exploited. Therefore, this work proposes a novel methodological approach involving the integration of several heterogeneous skin cancer series, and a later multiclass classifier design. This approach is thus a way to provide the clinicians with an intelligent diagnosis support tool based on the use of a robust set of selected biomarkers, which simultaneously distinguishes among different cancer-related skin states. To achieve this, a multi-platform combination of microarray datasets from Affymetrix and Illumina manufacturers was carried out. This integration is expected to strengthen the statistical robustness of the study as well as the finding of highly-reliable skin cancer biomarkers. Specifically, the designed operation pipeline has allowed the identification of a small subset of 17 differentially expressed genes (DEGs) from which to distinguish among 7 involved skin states. These genes were obtained from the assessment of a number of potential batch effects on the gene expression data. The biological interpretation of these genes was inspected in the specific literature to understand their underlying information in relation to skin cancer. Finally, in order to assess their possible effectiveness in cancer diagnosis, a cross-validation Support Vector Machines (SVM)-based classification including feature ranking was performed. The accuracy attained exceeded the 92% in overall recognition of the 7 different cancer-related skin states. The proposed integration scheme is expected to allow the co-integration with other state-of-the-art technologies such as RNA-seq.

Genome-scale examination of NBS-encoding genes in blueberry.

Blueberry is an important crop worldwide. It is, however, susceptible to a variety of diseases, which can lead to losses in yield and fruit quality. Although screening studies have identified resistant germplasm for some important diseases, still little is known about the molecular basis underlying that resistance. The most predominant type of resistance (R) genes contains nucleotide binding site and leucine rich repeat (NBS-LRR) domains. The identification and characterization of such a gene family in blueberry would enhance the foundation of knowledge needed for its genetic improvement. In this study, we searched for and found a total of 106 NBS-encoding genes (including 97 NBS-LRR) in the current blueberry genome. The NBS genes were grouped into eleven distinct classes based on their domain architecture. More than 22% of the NBS genes are present in clusters. Ten genes were mapped onto seven linkage groups. Phylogenetic analysis grouped these genes into two major clusters based on their structural variation, the first cluster having toll and interleukin-1 like receptor (TIR) domains and most of the second cluster containing a coiled-coil domain. Our study provides new insight into the NBS gene family in blueberry and is an important resource for the identification of functional R-genes.

Integration of RNA-Seq data with heterogeneous microarray data for breast cancer profiling.

BACKGROUND: Nowadays, many public repositories containing large microarray gene expression datasets are available. However, the problem lies in the fact that microarray technology are less powerful and accurate than more recent Next Generation Sequencing technologies, such as RNA-Seq. In any case, information from microarrays is truthful and robust, thus it can be exploited through the integration of microarray data with RNA-Seq data. Additionally, information extraction and acquisition of large number of samples in RNA-Seq still entails very high costs in terms of time and computational resources.This paper proposes a new model to find the gene signature of breast cancer cell lines through the integration of heterogeneous data from different breast cancer datasets, obtained from microarray and RNA-Seq technologies. Consequently, data integration is expected to provide a more robust statistical significance to the results obtained. Finally, a classification method is proposed in order to test the robustness of the Differentially Expressed Genes when unseen data is presented for diagnosis. RESULTS: The proposed data integration allows analyzing gene expression samples coming from different technologies. The most significant genes of the whole integrated data were obtained through the intersection of the three gene sets, corresponding to the identified expressed genes within the microarray data itself, within the RNA-Seq data itself, and within the integrated data from both technologies. This intersection reveals 98 possible technology-independent biomarkers. Two different heterogeneous datasets were distinguished for the classification tasks: a training dataset for gene expression identification and classifier validation, and a test dataset with unseen data for testing the classifier. Both of them achieved great classification accuracies, therefore confirming the validity of the obtained set of genes as possible biomarkers for breast cancer. Through a feature selection process, a final small subset made up by six genes was considered for breast cancer diagnosis. CONCLUSIONS: This work proposes a novel data integration stage in the traditional gene expression analysis pipeline through the combination of heterogeneous data from microarrays and RNA-Seq technologies. Available samples have been successfully classified using a subset of six genes obtained by a feature selection method. Consequently, a new classification and diagnosis tool was built and its performance was validated using previously unseen samples.

Design and Sampling Plan Optimization for RT-qPCR Experiments in Plants: A Case Study in Blueberry.

The qPCR assay has become a routine technology in plant biotechnology and agricultural research. It is unlikely to be technically improved, but there are still challenges which center around minimizing the variability in results and transparency when reporting technical data in support of the conclusions of a study. There are a number of aspects of the pre- and post-assay workflow that contribute to variability of results. Here, through the study of the introduction of error in qPCR measurements at different stages of the workflow, we describe the most important causes of technical variability in a case study using blueberry. In this study, we found that the stage for which increasing the number of replicates would be the most beneficial depends on the tissue used. For example, we would recommend the use of more RT replicates when working with leaf tissue, while the use of more sampling (RNA extraction) replicates would be recommended when working with stems or fruits to obtain the most optimal results. The use of more qPCR replicates provides the least benefit as it is the most reproducible step. By knowing the distribution of error over an entire experiment and the costs at each step, we have developed a script to identify the optimal sampling plan within the limits of a given budget. These findings should help plant scientists improve the design of qPCR experiments and refine their laboratory practices in order to conduct qPCR assays in a more reliable-manner to produce more consistent and reproducible data.

Expression Profile of Carotenoid Cleavage Dioxygenase Genes in Summer Squash (Cucurbita pepo L.).

Carotenoids are important dietary components that can be found in vegetable crops. The accumulation of these compounds in fruit and vegetables is altered by the activity of carotenoid cleavage dioxygenases (CCDs) enzymes that produce their degradation. The aim of this work was to study the possible implication of CCD genes in preventing carotenoid storage in the horticultural crop summer squash (Cucurbita pepo L.). The relationship between the presence of these compounds and gene expression for CCDs was studied in three varieties showing different peel and flesh colour. Expression analysis for the CCD genes CpNCED1, CpNCED2, CpNCED3, CpNCED9, CpCCD1, CpCCD4a, CpCCD4b and CpCCD8 was carried out on different organs and at several fruit developmental stages. The results showed that the CpCCD4a and CpCCD4b genes were highly expressed in the variety with lowest carotenoid content suggesting a putative role in carotenoid accumulation pattern in summer squash fruit.

First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement.

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.

Identification of Risk Factors for the Occurrence of Cement Leakage During Percutaneous Vertebroplasty for Painful Osteoporotic or Malignant Vertebral Fracture.

STUDY DESIGN: Retrospective study. OBJECTIVE: To identify fracture morphology-dependent risk factors for the occurrence of cement leakage during percutaneous vertebroplasty. SUMMARY OF BACKGROUND DATA: Extravertebral cement leakage is the most frequently reported complication and represents a major risk after vertebroplasty and balloon kyphoplasty. Despite the frequent occurrence of cement leakage and potentially dangerous complications and sequelae, there are few reports that examine its predictors. METHODS: Retrospective review of 194 consecutive patients who underwent percutaneous vertebroplasty for painful osteoporotic or malignant vertebral fracture. The influences of several parameters that might affect the occurrence of cement leakage were assessed using univariate and multivariate analyses. Cement leakage was assessed using computed tomography scanning and classified into 4 different types with different potential sequelae: through the basivertebral vein, through the segmental vein, through a cortical defect, and intradiscal leakage. RESULTS: Leakage of cement was detected in 209 of the 272 treated vertebrae (76.83%). The most common types of leakage detected were through the basivertebral vein (43.38%) and the segmental vein (42.27%). None of the evaluated variables showed a statistically significant effect. Only the absence of Kummell disease (P = 0.063) and a lower severity grade of collapse (P = 0.068) approached statistical significance. For basivertebral vein leakages, the location at the thoracolumbar level and the absence of Kummell disease (P < 0.05) were strong predictive factors for cement leakages. For segmental vein leakages, the odds decreased as the severity grade of collapse increased (P = 0.008). CONCLUSION: Each different vertebral fracture pattern has its own risk factors for cement leakage. For certain types of leakage, Kummell avascular necrosis as a protective factor and fracture severity grade with paradoxical effect have each demonstrated influence in the occurrence of cement leakage. LEVEL OF EVIDENCE: 4.

Helichrysum and grapefruit extracts inhibit carbohydrate digestion and absorption, improving postprandial glucose levels and hyperinsulinemia in rats.

Several plant extracts rich in flavonoids have been reported to improve hyperglycemia by inhibiting digestive enzyme activities and SGLT1-mediated glucose uptake. In this study, helichrysum ( Helichrysum italicum ) and grapefruit ( Citrus × paradisi ) extracts inhibited in vitro enzyme activities. The helichrysum extract showed higher inhibitory activity of α-glucosidase (IC50 = 0.19 mg/mL) than α-amylase (IC50 = 0.83 mg/mL), whereas the grapefruit extract presented similar α-amylase and α-glucosidase inhibitory activities (IC50 = 0.42 mg/mL and IC50 = 0.41 mg/mL, respectively). Both extracts reduced maltose digestion in noneverted intestinal sacs (57% with helichrysum and 46% with grapefruit). Likewise, both extracts inhibited SGLT1-mediated methylglucoside uptake in Caco-2 cells in the presence of Na(+) (56% of inhibition with helichrysum and 54% with grapefruit). In vivo studies demonstrated that helichrysum decreased blood glucose levels after an oral maltose tolerance test (OMTT), and both extracts reduced postprandial glucose levels after the oral starch tolerance test (OSTT). Finally, both extracts improved hyperinsulinemia (31% with helichrysum and 50% with grapefruit) and HOMA index (47% with helichrysum and 54% with grapefruit) in a dietary model of insulin resistance in rats. In summary, helichrysum and grapefruit extracts improve postprandial glycemic control in rats, possibly by inhibiting α-glucosidase and α-amylase enzyme activities and decreasing SGLT1-mediated glucose uptake.

Carotenogenic gene expression and carotenoid accumulation in three varieties of Cucurbita pepo during fruit development.

The control of gene expression is a crucial regulatory mechanism in carotenoid accumulation of fruits and flowers. We investigated the role of transcriptional regulation of nine genes involved in the carotenoid biosynthesis pathway in three varieties of Cucurbita pepo with evident differences in fruit color. The transcriptional levels of the key genes involved in the carotenoid biosynthesis were higher in flower-, leaf-, and fruit skin tissues than flesh tissues. This correlated with higher concentration of carotenoid content in these tissues. The differential expression among the colored and white cultivars detected for some genes, such as LCYe, in combination with other regulatory mechanisms, could explain the large differences found in terms of carotenoid content among the three varieties. These results are a first step to elucidate carotenogenesis in C. pepo and demonstrate that, in general, regulation of the pathway genes is a critical factor that determines the accumulation of these compounds.

In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes.

A wide variety of qPCR methods currently exist for Salmonella spp., Escherichia coli O157 and Listeria monocytogenes detection. These methods target several genes and use different detection chemistries, either in simplex or in multiplex formats. However, the majority of these methods have not been carefully validated, and the number of validated methods that use multiplex qPCR is even lower. The aim of the present study was to develop and validate a multiplex qPCR method from previously validated simplex qPCR primers and probes. A modified broth medium was selected and primary and secondary enrichment times were further optimized. Efficiency of the newly combined qPCR system was comprised between 91% and 108%, for simplex and multiplex analyses. A total of 152 food and environmental, natural and spiked samples, were analyzed for the evaluation of the method obtaining values above 91% that were reached for all the quality parameters analyzed. A very low limit of detection (5 cfu/25 g after enrichment) for simultaneous identification of these 3 pathogens was obtained.

Screening of polyphenolic plant extracts for anti-obesity properties in Wistar rats.

BACKGROUND: Polyphenols have been reported to prevent chronic diseases such as cardiovascular diseases, cancers, diabetes and neurodegenerative diseases. The objective of the study was to conduct a screening for potential anti-obesity polyphenolic plant extracts using a diet-induced animal model. Rats were fed a high-fat-sucrose (HFS) diet with or without supplementation of different polyphenolic plant extracts (almond, apple, cinnamon, orange blossom, hamamelis, lime blossom, grape vine, and birch) for 56-64 days. RESULTS: Body weight gain was lower in rats supplemented with apple, cinnamon, hamamelis and birch extracts as compared to HFS non-supplemented group. Moreover, apple and cinnamon extracts prevented the increase in fat mass promoted by the HFS diet. Insulin resistance, estimated by the homostatic model assessment-insulin resistance (HOMA-IR) index, was reduced in rats fed apple, cinnamon, hamamelis and birch extracts. Apple extract also prevented the HFS-induced hyperglycaemia and hyperleptinaemia. CONCLUSION: Only apple and cinnamon extracts were finally considered as potentially important anti-obesogenic extracts, due to their body fat-lowering effects, while the improvement of obesity-related metabolic complications by apple polyphenols highlights this extract as a promising functional food ingredient for the management of obesity and its metabolic complications.

RNA quality assessment: a view from plant qPCR studies.

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is probably the most common molecular technique used in transcriptome analyses today. The simplicity of the technology and associated protocols that generate results without the need to understand the underlying principles has made RT-qPCR the method of choice for RNA quantification. Rather than the 'gold standard technology' often used to describe it, the performance of RT-qPCR suffers from considerable pitfalls during general workflow. The inconsistency of conventional methods for the evaluation of RNA quality and its influence on qPCR performance as well as stability of reference genes is summarized and discussed here.

Evaluation of different pulverisation methods for RNA extraction in squash fruit: lyophilisation, cryogenic mill and mortar grinding.

INTRODUCTION: Quality and integrity of RNA are critical for transcription studies in plant molecular biology. In squash fruit and other high water content crops, the grinding of tissue with mortar and pestle in liquid nitrogen fails to produce a homogeneous and fine powered sample desirable to ensure a good penetration of the extraction reagent. OBJECTIVE: To develop an improved pulverisation method to facilitate the homogenisation process of squash fruit tissue prior to RNA extraction without reducing quality and yield of the extracted RNA. METHODOLOGY: Three methods of pulverisation, each followed by the same extraction protocol, were compared. The first approach consisted of the lyophilisation of the sample in order to remove the excess of water before grinding, the second one used a cryogenic mill and the control one a mortar grinding of frozen tissue. The quality of the isolated RNA was tested by carrying out a quantitative real time downstream amplification. RESULTS: In the three situations considered, mean values for A(260) /A(280) indicated minimal interference by proteins and RNA quality indicator (RQI) values were considered appropriate for quantitative real-time polymerase chain reaction (qRT-PCR) amplification. Successful qRT-PCR amplifications were obtained with cDNA isolated with the three protocols. CONCLUSION: Both apparatus can improve and facilitate the grinding step in the RNA extraction process in zucchini, resulting in isolated RNA of high quality and integrity as revealed by qRT-PCR downstream application. This is apparently the first time that a cryogenic mill has been used to prepare fruit samples for RNA extraction, thereby improving the sampling strategy because the fine powder obtained represents a homogeneous mix of the organ tissue.

Characterization of the 3':5' ratio for reliable determination of RNA quality.

Determination of RNA quality is a critical first step in obtaining meaningful gene expression data. The PCR-based 3':5' assay is an RNA quality assessment tool. This assay is a simple, fast, and low-cost method of selecting samples for further analysis. However, its practical applications are unexploited primarily because of the absence of an experimental threshold. We show that, by anchoring the 5' assay a specific distance from the 3' end of the sequence and by spacing the 3' at a distance of a number of nucleotides, a cutoff determines whether a sample is suitable for downstream quantification studies.

Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR.

The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop.

Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions.

Reverse transcription quantitative real-time polymerase chain reaction is the most accurate measure of gene expression in biological systems. The data are analyzed through a process called normalization. Internal standards are essential for determining the relative gene expression in different samples. For this purpose, reference genes are selected based on their constitutive expression across samples. At present, there has not yet been any reference gene identified in any organism that is universally optimal across different tissue types or disease situations. Our goal was to test the regulation of 11 potential references for pea. These included eight commonly used and three new candidates. Twenty-six samples, including different tissues, treatments and genotypes, were addressed in this analysis. For reliable data normalization, the most suitable combination of reference genes in each experimental set was constructed with at least two out the five more stably expressed references in the whole experimental series (i.e. protein phosphatase 2A, beta-tubulin, GH720838, actin and GH720808). To validate the determined measure of gene-stability, the gene-specific variation was calculated using different normalization factors. The most non-specific variation was removed when the most stable genes were used, highlighting the importance of the adequate choice of internal controls in gene expression experiments. The set of reference genes presented here will provide useful guidelines as starting point for reference gene selection in pea studies under conditions other than those tested here.

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