The anteriorcruciateligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Geneexpression analysis may be a useful tool for under- standing ACL tears and healing failure. Reversetranscription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use ofsuitablereferencegenes for data normalization. Here, we evaluated the suitability of six referencegenes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) byusing 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 referencegenes was determined byusing the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single referencegene and ACTB+TBP was the best gene pair. The GenEx soft- ware showed that the accumulated standard deviation is reduced when a larger number ofreferencegenes is used for geneexpression normalization. However, the use of a single referencegene may not be suitable. To identify the optimal combination ofreferencegenes, we evaluated the expressionof FN1 and PLOD1. We observed that at least 3 referencegenes 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 geneexpression 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 referencegenes for analysis of ACL samples of individuals with and without ACL tears.
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-transcriptionquantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use ofsuitablereferencegenes 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 referencegenes was determined using the NormFinder, geN- orm, BestKeeper and DataAssist software packages. Overall, HPRT1 was the best single referencegene, and HPRT1+TBP composed the best pair and HPRT1+TBP+ACTB com- posed the best trio ofreferencegenes from the analysis of different groups, including the si- multaneous analysis of all tissue samples. To identify the optimal combination ofreferencegenes, we evaluated the expressionof COL1A1 and COL3A1, and no obvious differences were observed when using 2, 3 or 4 referencegenes 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 ofreferencegenes (HPRT1+TBP+ACTB) and 4 referencegenes did not revealed a significant difference in COL3A1 expression. Consequently, the use ofsuitablereferencegenes for a reliable geneexpression evaluation by RT-qPCR should consider the type of tendon samples in- vestigated. HPRT1+TBP+ACTB seems to be the best combination ofreferencegenes for the analysis of involving different tendon samples of individuals with rotator cuff tears.
Methods for the quantification of accurate gene expres- sion have an increasingly important role in studies aiming for the reliable examination ofexpression profiles gener- ated by high-throughput approaches. Real-time reversetranscriptionquantitativePCR (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 referencegene, 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 referencegene should be used to avoid misinterpretation ofgeneexpression data [6,7,11-13].
Shoulder instability is a common shoulder injury, and patients present with plastic deformation of the glenohumeral capsule. Geneexpression analysis may be a useful tool for increasing the general understanding of capsule deformation, and reverse-transcriptionquantitative 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 ofsuitablereferencegenes for data normalization to guarantee meaningful and reproducible results. In the present study, we evaluated the suitability of a set ofreferencegenesusing samples from the glenohumeral capsules of individuals with and without shoulder instability. We analyzed the expressionof six commonly used referencegenes (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 referencegeneexpression was determined using four software packages: NormFinder, geNorm, BestKeeper and DataAssist. Overall, HPRT1 was the best single referencegene, and HPRT1 and B2M composed the best pair ofreferencegenes from different analysis groups, including simultaneous analysis of all tissue samples. GenEx software was used to identify the optimal number ofreferencegenes to be used for normalization and demonstrated that the accumulated standard deviation resulting from the use of 2 referencegenes was similar to that resulting from the use of 3 or more referencegenes. To identify the optimal combination ofreferencegenes, we evaluated the expressionof COL1A1. Although the use of different referencegene 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 referencegenes. Consequently, the use of 2 stable referencegenes for normalization, especially HPRT1 and B2M, is a reliable method for evaluating geneexpressionby RT-qPCR.
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 . Jujube whole-genome sequencing has recently been completed , and gene function analysis, which is an important aspect of fol- low-up studies, requires the appropriate selection ofreferencegenes. For jujube, ZjH3 is report- edly the most suitablegene for evaluating geneexpressionin early-growth fruit-bearing shoots, shoot apices, and different organs by semi-quantitativereversetranscriptionPCR (RT-PCR) , and recently UBQ, ACTIN9, UBQ2 and CYP were validated as stable genes at some restricted conditions byreversetranscription-quantitative real-time polymerase chain reaction (RT-qPCR) . The identification and validation of additional jujube referencegenesby RT- qPCR will further contribute to related studies at the transcription level.
Geneexpression analysis plays a major role in understanding of the complex regulatory net- works and identificationofgenes relevant to new biological processes . Among several tech- niques available for transcript level measurement, real time quantitativereversetranscriptionPCR (qRT-PCR) is widely used due to its specificity, sensitivity and dynamic range . Valida- tion of high-throughput technologies such as RNA-sequencing (RNA-seq) through qRT-PCR further implies the robustness of this technique . 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 byusing proper controls . Applying the internal control gene known as referencegene 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 referencegene is expected to be stable in terms ofexpression level across various conditions such as develop- mental stages, organ types, and experimental conditions . The most frequently used refer- ence genes are the 18S ribosomal RNA (18SrRNA), glyceraldehyde-3-phosphate
Many candidate referencegenes, such as actin, a-tubulin, translation elongation factor, and ADP-ribosylation factor, belong to different families. Genesin the same family have conserved sequences. In the pre-genomic era, it was hard to design primers to specifically amplify one genein a family because there was a dearth of sequence information regarding other members. This may cause simultaneous amplification of two or more genesin the same family when the primer pair is located in the conserved region. Different genesin the same family usually have different expression patterns. In wheat, the stability ofexpressionof 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 . 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 genein a gene family for use as candidate reference is a very good strategy when the genome sequence is available.
Real-time quantitativereversetranscription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns ofgeneexpression. The reliability of qPCR is highly dependent on the selection of appropriate referencegenes used for normalization. By analyzing 16 potential candidates ofreferencegenes (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 referencegenes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expressionof Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reversetranscription efficiency, we recommended the use of Ppia and Hprt as the most suitablegenes 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 referencegenes 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.
posterior cruciateligament. 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.
Studies conducted on sugarcane to determine referencegenes 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 geneexpressionin the roots, and GUO et al. (2014) analyzed geneexpressionin 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 referencegenes 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 referencegenes 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).
Venipuncture is one of the easiest clinical procedures to obtain viable blood samples to evaluate geneexpressionusing mRNA analysis. However, the use of this sample type inreverse 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 geneexpressionusing Real Time PCR.
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 AnteriorCruciateLigament - 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.
Referencegenes are commonly used for normalization of target geneexpression during RT-qPCR analysis. How- ever, no housekeeping genes or referencegenes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitablereferencegenes for RT-qPCR analysis of target geneexpressionin the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, wa- ter temperature, and drug treatments), seven referencegenes, 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 ofgeneexpression 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 ofquantitative analysis ofgeneexpression. The findings highlighted the importance of validation of housekeeping genes for research on geneexpression under different conditions of experiment and species.
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 genesin 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 expressionof 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.
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.
Medical records of patients with a uniform criteria di- agnosis ofligamentinjuryof 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 ).
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 expressionof 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 expressionof SF-1 during testis development, and results in the premature commitment of progenitor cells to the steroidogenic lineage because of the expressionof Sf-1 target genes (13). Thus, there is strong evidence to suggest that POD-1/Tcf21 is involved in the regulation of SF-1 expressionin the testis; however, it is unclear whether POD-1/Tcf21 plays a role in the control of SF-1 expressionin the adrenal cortex.
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 anteriorcruciateligament 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-
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, Roditis 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 geneof 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.
With the aim of finding suitablereferencegenes for geneexpression 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 PCRingeneexpression studies on endometrial cancer tissues. Within these reports, we removed papers evaluating microRNA expression and genotyping studies as well as geneexpression 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 geneexpression studies in fresh frozen tissues were used in the final analysis. These articles used the following referencegenes 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),