Top PDF Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for under- standing ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx soft- ware showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving iso- lated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three indepen- dent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.
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Identification of Suitable Reference Genes for Gene Expression Studies in Tendons from Patients with Rotator Cuff Tear

Identification of Suitable Reference Genes for Gene Expression Studies in Tendons from Patients with Rotator Cuff Tear

Rotator cuff tear is one of the most common causes of shoulder dysfunction. Gene expres- sion analysis may be a useful tool for understanding tendon tears and the failure of cuff healing, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluate the suitability of six ref- erence genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using samples from the rotator cuff tendons of 28 individuals with tendon tears (3 tendons regions) and 8 controls (2 tendon regions); for the tear patients, we evaluated ruptured and non-ruptured tendon samples. The stability of the candidate reference genes was determined using the NormFinder, geN- orm, BestKeeper and DataAssist software packages. Overall, HPRT1 was the best single reference gene, and HPRT1+TBP composed the best pair and HPRT1+TBP+ACTB com- posed the best trio of reference genes from the analysis of different groups, including the si- multaneous analysis of all tissue samples. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1 and COL3A1, and no obvious differences were observed when using 2, 3 or 4 reference genes for most of the analyses. However, COL3A1 expression differed between ruptured and non-ruptured (posterior superior region) tendons of patients only when normalized by HPRT1+TBP+B2M and HPRT1+TBP. On the other hand, the comparison between these two groups using the best trio of reference genes (HPRT1+TBP+ACTB) and 4 reference genes did not revealed a significant difference in COL3A1 expression. Consequently, the use of suitable reference genes for a reliable gene expression evaluation by RT-qPCR should consider the type of tendon samples in- vestigated. HPRT1+TBP+ACTB seems to be the best combination of reference genes for the analysis of involving different tendon samples of individuals with rotator cuff tears.
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Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Methods for the quantification of accurate gene expres- sion have an increasingly important role in studies aiming for the reliable examination of expression profiles gener- ated by high-throughput approaches. Real-time reverse transcription quantitative PCR (qRT-PCR) has emerged as one of the most powerful tools for this purpose. Given the extreme sensitivity of qRT-PCR, a careful and stringent selection of a proper constitutively expressed control gene is required to account for differences in the amount and quality of starting RNA and in cDNA synthesis efficiency. Adequate normalizations presume the use of an internal control, often referred to as a housekeeping or reference gene, whose expression levels should not significantly vary among tissues and experimental situations analyzed [1,2]. Genes most commonly applied as references in qRT-PCR studies include: beta actin (ACTB), glyceral- deyde-3-phosphate dehydrogenase (GAPDH), beta glu- curonidase (GUSB), hypoxanthine guanine phosphoribosyl transferase (HPRT1) and ribosome small subunit (18S) ribosomal RNA [1-3]. However, several reports have mentioned these classical housekeeping genes as showing variable expression levels in different experimental conditions [3-9]. Furthermore, the same gene revealed as almost invariant for certain tissues or cell types or could present highly variable expression levels in other tissues or experimental conditions [2,9,10]. Thus, it is clear that suitable control genes are extremely specific for particular sample sets and experimental models, being a crucial component in assessing confident gene expres- sion patterns. It has been strongly suggested that more than one stable expressed reference gene should be used to avoid misinterpretation of gene expression data [6,7,11-13].
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Identification of Suitable Reference Genes for Gene Expression Studies of Shoulder Instability

Identification of Suitable Reference Genes for Gene Expression Studies of Shoulder Instability

Shoulder instability is a common shoulder injury, and patients present with plastic deformation of the glenohumeral capsule. Gene expression analysis may be a useful tool for increasing the general understanding of capsule deformation, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. Although RT-qPCR is highly sensitive and specific, it requires the use of suitable reference genes for data normalization to guarantee meaningful and reproducible results. In the present study, we evaluated the suitability of a set of reference genes using samples from the glenohumeral capsules of individuals with and without shoulder instability. We analyzed the expression of six commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, TBP and TFRC) in the antero- inferior, antero-superior and posterior portions of the glenohumeral capsules of cases and controls. The stability of the candidate reference gene expression was determined using four software packages: NormFinder, geNorm, BestKeeper and DataAssist. Overall, HPRT1 was the best single reference gene, and HPRT1 and B2M composed the best pair of reference genes from different analysis groups, including simultaneous analysis of all tissue samples. GenEx software was used to identify the optimal number of reference genes to be used for normalization and demonstrated that the accumulated standard deviation resulting from the use of 2 reference genes was similar to that resulting from the use of 3 or more reference genes. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1. Although the use of different reference gene combinations yielded variable normalized quantities, the relative quantities within sample groups were similar and confirmed that no obvious differences were observed when using 2, 3 or 4 reference genes. Consequently, the use of 2 stable reference genes for normalization, especially HPRT1 and B2M, is a reliable method for evaluating gene expression by RT-qPCR.
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Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.

Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.

Chinese jujube (Ziziphus jujuba Mill.), a member of the genus Ziziphus in the family Rhamna- ceae, is widely distributed in temperate and subtropical areas of the Northern Hemisphere, especially the inland region of Northern China [1]. Jujube whole-genome sequencing has recently been completed [2], and gene function analysis, which is an important aspect of fol- low-up studies, requires the appropriate selection of reference genes. For jujube, ZjH3 is report- edly the most suitable gene for evaluating gene expression in early-growth fruit-bearing shoots, shoot apices, and different organs by semi-quantitative reverse transcription PCR (RT-PCR) [3], and recently UBQ, ACTIN9, UBQ2 and CYP were validated as stable genes at some restricted conditions by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) [4]. The identification and validation of additional jujube reference genes by RT- qPCR will further contribute to related studies at the transcription level.
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Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

Gene expression analysis plays a major role in understanding of the complex regulatory net- works and identification of genes relevant to new biological processes [3]. Among several tech- niques available for transcript level measurement, real time quantitative reverse transcription PCR (qRT-PCR) is widely used due to its specificity, sensitivity and dynamic range [4]. Valida- tion of high-throughput technologies such as RNA-sequencing (RNA-seq) through qRT-PCR further implies the robustness of this technique [5]. However, several factors including the quantity and integrity of the extracted RNA, the efficiencies of cDNA synthesis and the prod- ucts of polymerase chain reaction (PCR) can significantly generate non-specific variations, which should be corrected by using proper controls [6]. Applying the internal control gene known as reference gene or housekeeping gene is the most common way to normalize the tran- script level and reduce the inherent experimental errors [6–8]. Therefore, the normalization is a crucial step before performing any qRT-PCR analysis. Ideally, an appropriate reference gene is expected to be stable in terms of expression level across various conditions such as develop- mental stages, organ types, and experimental conditions [9]. The most frequently used refer- ence genes are the 18S ribosomal RNA (18SrRNA), glyceraldehyde-3-phosphate
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Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

Many candidate reference genes, such as actin, a-tubulin, translation elongation factor, and ADP-ribosylation factor, belong to different families. Genes in the same family have conserved sequences. In the pre-genomic era, it was hard to design primers to specifically amplify one gene in a family because there was a dearth of sequence information regarding other members. This may cause simultaneous amplification of two or more genes in the same family when the primer pair is located in the conserved region. Different genes in the same family usually have different expression patterns. In wheat, the stability of expression of multi- transcript targeting references from the same family and single sequence amplification were compared in the gene families of actin, tubulin, EF, and ADP. The results proved that primer pairs targeting single gene for each of the analyzed families performed better than those targeting multiple genes [35]. In the present study, the melon genome information was used to design specific primers to each candidate, and genes from the same family were also evaluated for their expression stability. CmACT (b-actin) exhibited better expression stability than CmACT7 as determined by both geNorm and NormFinder. CmRPL showed better performance than CmRPL2. These results show that using specific primer pair to amplify a single gene in a gene family for use as candidate reference is a very good strategy when the genome sequence is available.
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Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.

Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.

Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.
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Rev. bras. ortop.  vol.46 número2 en a05v46n2

Rev. bras. ortop. vol.46 número2 en a05v46n2

posterior cruciate ligament. The cannula of the tibial guide is placed at a 45º angle to the longitudinal axis of the tibia, and the angle between the initial point of the tibia tunnel and the horizontal arm of the guide is fixed at 55º, to produce an ideal orientation for the transtibial femoral perforation. With the guide in po- sition, the tibia is perforated with a Kirshner wire, and a reamer with equal diameter to that of the tendinous graft makes the tibial tunnel, guided by the intraos- seous wire. The femoral tunnel is prepared with the knee flexed at 90°. We used a transtibial guide, posi- tioned on the medial side of the lateral condyle of the femur at 10 o’clock for the left knee and at 2 o’clock for the right knee, 2 mm from the posterior cortical wall. With the guide in position, the femur is perfo- rated with a Kirshner wire, which guides a reamer of the same diameter as the tendinous graft, producing a femoral tunnel of 3.5 cm in depth.
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Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars

Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars

Studies conducted on sugarcane to determine reference genes have been conducted in plants grown in pots (SILVA et al. 2014), in vitro with Saccharum officinarum (LING et al. 2014), and in the field (ISKANDAR et al. 2004). Nevertheless, only SILVA et al. (2014) determined stability associated with drought stress in plants in glasshouse pots, analyzing gene expression in the roots, and GUO et al. (2014) analyzed gene expression in seedlings by inducing drought stress with polyethylene glycol 8000 (PEG). Neither study included gene stability analysis in field-grown adult plants. In sugarcane, the most commonly used reference genes are: 25S ribosomal ribonucleic acid (rRNA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, γ-actin, α-tubulin, β-tubulin, and ubiquitin, among others. In this study, eight genes: ubiquitin (Ubi), α-tubulin (α –tub), β-tubulin (β-tub), β-actin (β-act), GAPDH, histone H3 (ht3), eukaryotic elongation factor 1α (eEF1) and cyclophilin (Cyc) were evaluated as reference genes for drought stress in sugarcane leaves under field conditions of drought stress (SILVA et al. 2014; GUO et al. 2014; RODRIGUES et al. 2011; ANDRADE et al. 2017).
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Rev. Bras. Hematol. Hemoter.  vol.32 número5

Rev. Bras. Hematol. Hemoter. vol.32 número5

Venipuncture is one of the easiest clinical procedures to obtain viable blood samples to evaluate gene expression using mRNA analysis. However, the use of this sample type in reverse transcriptase polymerase chain reaction tests (RT-PCR) without prior treatment is controversial. We therefore propose to compare the suitability of different peripheral blood samples (whole blood without treatment, whole blood with hemolysis, peripheral blood mononuclear cells and frozen whole blood) for RT-PCR analysis. The results showed that, despite the blood sample being peripheral, it is possible to extract a fair amount of RNA and perform target gene amplification. Thus, peripheral blood without prior treatment could be used to investigate the gene expression using Real Time PCR.
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Repositório Institucional UFC: Retorno ao esporte no nível pré-lesão após reconstrução do ligamento cruzado anterior: fatores preditores

Repositório Institucional UFC: Retorno ao esporte no nível pré-lesão após reconstrução do ligamento cruzado anterior: fatores preditores

Introdução: A lesão do ligamento cruzado anterior (LCA) está entre as condições de joelho mais prevalentes, ocasionando diversos déficits funcionais. A reconstrução do LCA (R-LCA) é um procedimento comum para o retorno à prática esportiva. Objetivos: Verificar os fatores preditores de retorno ao esporte no mesmo nível pré- lesão e suas interações em indivíduos que realizaram R-LCA. Metodologia: Trata-se de um estudo transversal composto por 136 participantes, com no mínimo 6 meses após de R-LCA. Todos os participantes responderam os questionários International Knee Documentation Committee (IKDC) e Anterior Cruciate Ligament - Return to Sport After Injury (ACL-RSI) e realizaram avaliação de estabilidade postural no Biodex Balance System e de força dos músculos quadríceps e isquiotibiais por meio do Dinamômetro Isocinético. A Árvore de Classificação e Regressão (CART) foi utilizada para estabelecer uma relação entre os vetores de variáveis preditoras e a variável resposta e a Odds Ratio para verificar a probabilidade de não retorno no nível pré-lesão. Resultados: A variável preditora mais forte foi o ACL-RSI, com nota de corte de 72,85%. Como fatores secundários, obtivemos o índice de estabilidade global do membro lesionado, índice de simetria entre membros para extensão e IKDC, com notas de corte de 3,2º, 16,6% e 82,75% respectivamente. O modelo é capaz de verificar em 60% das vezes quem não consegue retornar, com especificidade de 97,4%. Conclusão: O componente psicológico, observado pelo ACL-RSI, é o fator preditor mais importante para o retorno ao esporte no nível pré-lesão. Secundariamente, podemos considerar estabilidade global do membro lesionado, força simétrica para extensão e função, relatada pelo IKDC.
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Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. How- ever, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, wa- ter temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experi- mental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best can- didate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.
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Loss of function in Mlo orthologs reduces susceptibility of pepper and tomato to powdery mildew disease caused by Leveillula taurica.

Loss of function in Mlo orthologs reduces susceptibility of pepper and tomato to powdery mildew disease caused by Leveillula taurica.

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse tomato and pepper production. In contrast to most powdery mildew species which are epiphytic, L. taurica is an endophytic fungus colonizing the mesophyll tissues of the leaf. In barley, Arabidopsis, tomato and pea, the correct functioning of specific homologues of the plant Mlo gene family has been found to be required for pathogenesis of epiphytic powdery mildew fungi. The aim of this study was to investigate the involvement of the Mlo genes in susceptibility to the endophytic fungus L. taurica. In tomato (Solanum lycopersicum), a loss-of-function mutation in the SlMlo1 gene results in resistance to powdery mildew disease caused by Oidium neolycopersici. When the tomato Slmlo1 mutant was inoculated with L. taurica in this study, it proved to be less susceptible compared to the control, S. lycopersicum cv. Moneymaker. Further, overexpression of SlMlo1 in the tomato Slmlo1 mutant enhanced susceptibility to L. taurica. In pepper, the CaMlo2 gene was isolated by applying a homology-based cloning approach. Compared to the previously identified CaMlo1 gene, the CaMlo2 gene is more similar to SlMlo1 as shown by phylogenetic analysis, and the expression of CaMlo2 is up-regulated at an earlier time point upon L. taurica infection. However, results of virus-induced gene silencing suggest that both CaMlo1 and CaMlo2 may be involved in the susceptibility of pepper to L. taurica. The fact that overexpression of CaMlo2 restored the susceptibility of the tomato Slmlo1 mutant to O. neolycopersici and increased its susceptibility to L. taurica confirmed the role of CaMlo2 acting as a susceptibility factor to different powdery mildews, though the role of CaMlo1 as a co-factor for susceptibility cannot be excluded.
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Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development

Nesse estudo nós usamos a técnica de Differential Dis- play Reverse Transcriptase – Polymerase Chain Reaction (DDRT-PCR) para comparamos o perfil de mRNA em Melipona scutellaris durante o desenvolvimento onto- genético pós-embrionário e em operárias adultas, rainha natural e induzida pelo Hormônio Juvenil III. Fragmen- tos diferencialmente expressos foram detectados usando as seguintes combinações de primers: HT11G-AP05; HT11C-AP05; HT11G-OPF12; HT11G-OPA16. Dos 9 ESTs descrito nesse trabalho, 6 tiveram expressão dife- rencial nas fases de larva L1 e L2, sugerindo serem meca- nismos chave no regulação do desenvolvimento larval em Melipona. A combinação HT11G-AP05 revelou em L1 e L2 um produto com similaridade à proteína tioredoxina redutase de Clostridium sporogenes, uma proteína impor- tante durante os processos de oxidoredução. Esse estudo representa as primeiras evidências moleculares do perfil de expressão durante o desenvolvimento ontogenético em abelhas do gênero Melipona.
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Clinics  vol.63 número1

Clinics vol.63 número1

Medical records of patients with a uniform criteria di- agnosis of ligament injury of the knee who were surgically treated at a knee pathology Reference Service between Oc- tober 2003 and December 2006 were reviewed. The preoperative evaluation of the patients included history and physical examination, standard radiographs and magnetic resonance imaging (MRI). A total of 978 patients were iden- tified. Of these, 109 presenting at least two associated liga- ment injuries in the same knee were eligible for the study. All injuries were chronic (over three weeks in duration 2 ).
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POD-1Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells

POD-1Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells

upstream stimulatory factors 1 and 2 (USF1/2) increase Sf-1 transcription through interactions with an E-box binding site spanning the  81/  76 region of the Sf-1 promoter (9,12-14). POD-1/capsulin/Tcf21 is another helix-loop-helix factor implicated in transcriptional regula- tion after binding to the E-box element of the Sf-1 promoter. However, in contrast to USF1/2, POD-1 represses the expression of Sf-1 in fetal mouse testis by preventing the binding of USF1/2 to the Sf-1 E-box sequence (13,15). Deletion of Pod-1 leads to increased expression of SF-1 during testis development, and results in the premature commitment of progenitor cells to the steroidogenic lineage because of the expression of Sf-1 target genes (13). Thus, there is strong evidence to suggest that POD-1/Tcf21 is involved in the regulation of SF-1 expression in the testis; however, it is unclear whether POD-1/Tcf21 plays a role in the control of SF-1 expression in the adrenal cortex.
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Basic principles of aggressive rehabilitation after anterior cruciate ligament reconstruction

Basic principles of aggressive rehabilitation after anterior cruciate ligament reconstruction

12. Mikkelsen S, Werner S, Erikkson F, et al. Closed kinetic chain alone compared to combined open and closed kinetic chain exer- cises for quadriceps strengthening after anterior cruciate ligament reconstruction with respect to return to sports: a prospective matched follow-up study. Am J Knee Surg 2000; 8(6):337-42. 13. Lysholm M, Ledin T, Odkvist M, et al. Postural control – a compar-

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Control of gene expression in Trypanosomatidae

Control of gene expression in Trypanosomatidae

transcribed at low levels in bloodstream para- sites indicating that some post-transcription mechanisms must take place in order to pre- vent the accumulation of PARP mRNA. Moreover, although PARP and PAG genes are derived from the same polycistronic tran- scription unit, there is a 100-fold difference in the steady-state levels when PARP and PAG mRNAs are compared (31). Several possibilities have been considered to explain this difference in the accumulation of mRNAs, including differences in trans-splic- ing efficiency and the presence of regulatory sequences in the 3'-untranslated regions (UTR) affecting the steady-state levels of these mRNAs. The study of regulatory ele- ments derived from the 3'UTR has been extensively explored (28,31,34,35). Using transient transfections, Roditi’s group (35) has demonstrated that a 16-mer sequence which is predicted to form a stem-loop struc- ture in the 3'UTR of PARP mRNAs can confer regulation of a CAT reporter gene on procyclics. Deletion of this element causes a 12-fold drop in CAT expression but no sig- nificant difference in the levels of total RNA, suggesting control at the translational level. Interestingly, the same sequence, with the same predicted secondary structure, has also been found in the analogous gene of another trypanosome species, T. congolense. More recently, a systematic analysis of the entire 297-base 3'UTR revealed three additional elements which are involved in post-tran- scriptional regulation of PARP genes, all three of them having an effect on the rate of mRNA turnover (34). It seems that, by em- ploying several layers of regulation, these parasites can ensure that the rapid changes associated with transmission between insect vector and mammalian host are followed by an instant reprogramming of genetic expres- sion.
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Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.

Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.

With the aim of finding suitable reference genes for gene expression studies in EC tissue samples, we carried out a Medline search using the MeSH terms ‘‘endometrial cancer’’ and ‘‘real-time PCR’’. We critically evaluated 327 papers published from May 2000 to February 2014 (see Text S1). We identified a total 102 articles based on the use of Real-Time PCR in gene expression studies on endometrial cancer tissues. Within these reports, we removed papers evaluating microRNA expression and genotyping studies as well as gene expression studies performed on cell cultures or Formalin-Fixed Paraffin Embedded (FFPE) tissues. Twelve additional articles were excluded from analysis because full text was not available in the English language. The remaining 90 articles focusing on gene expression studies in fresh frozen tissues were used in the final analysis. These articles used the following reference genes for data normalization, alone or in combination of two or three genes used in the final data analysis: Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) (39 times), Beta-actin (ACTB) (23 times), 18S-rRNA (22 times), hypoxanthine-guanine phosphoribosyltransferase (HPRT1) (6 times), peptidylprolyl isomerase A (PPIA) (5 times), DNA-directed RNA polymerase II subunit RPB1 (POLR2A) and b-glucuronidase (GUSB) (3 times each), ribosomal protein large P0 (RPLPO), ribosomal protein L19 (L19), and b2- microglobulin (B2M) (2 times each), TATA box binding protein (TBP),
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