The need to base qPCR analysis on dilution series data made relative quantification practically equivalent to the other flavor ofqRT-PCRanalysis, absolute quantification . Under this approach, the raw Cq (‘‘cycle of quantification’’) values are transformed into concentrations of the target per qRT-PCR reaction, based on the calibration curve constructed across a series of known target concentrations. The efficiency of amplification is implicitly taken into account during this conversion. Since in qRT- PCR the knowledge of the absolute target amount is usually not as important as the knowledge of its variation across samples, so- called relative calibration curves, created by diluting an arbitrary amount of target, are often used [12,13]. These relative calibration curves provide essentially the same information as the calibration curves for determining PCR efficiency . To account for variation in template loading across samples, the procedure called normalization is performed where the inferred target amounts are divided by the abundance of a control gene. In this way, all the target abundances become expressed as fold differences relative to the abundance of the control gene . However, hardly any one gene remains perfectly stable . Vandesompele et al  proposed that more accurate normalization could be achieved by using multiple nearly-stable controlgenes: in this case, the target abundances are divided by the geometric average of the control gene abundances. The same paper also introduced a non- parametric method, geNorm, for identification of the most stable controlgenes based on covariance across samples, which quickly became a standard in the qRT-PCR field . Another commonly used method for identifying stable genes is the parametric NormFinder algorithm , which makes use of the fact that the log-transformed qRT-PCRdata satisfy the normality criterion  and uses moments equations to calculate the stability of each gene independently of other genes. Following normaliza- tion, several authors used further parametric approaches such as ANOVA [6,19] and linear mixed models [20–23], applied on a gene-by-gene basis, to achieve maximum versatility in analysisof complicated designs. The workflow involving correction for amplification efficiency followed by multi-gene normalization and gene-by-gene analysis with t-tests, ANOVA, or linear modeling represents the current consensus ofqRT-PCRdata processing .
The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellu- lar development. Fruiting bodies of C. cinerea are typical mushrooms, which can be pro- duced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was per- formed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCRdata, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysisof DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.
To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1b, TBP, NADH, HSP22, GAPDH, Actin, a-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysisof the ten candidate reference genes identified a-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (a-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes a- Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.
In-gel tryptic digestion was performed as previously described . The peptides were then separated using an UltiMate nanoLC (LC Packings, Amsterdam) equipped with a PepMap C18 trap & column, using a gradient of increasing acetonitrile concentration, containing 0.1 % formic acid (5–35% acetonitrile in 180 min respectively, 35–50% in a further 30 min, followed by 95% acetonitrile to clean the column). The eluent was sprayed into a Q-Star XL tandem mass spectrometer (ABSciex, Foster City, CA) and analysed in Information Dependent Acquisition (IDA) mode, performing 1 sec of MS followed by 3 sec MSMS analyses of the 2 most intense peaks seen by MS. These masses are then excluded from analysis for the next 60 sec. MS/MS data for doubly and triply charged precursor ions was converted to centroid data, without smoothing, using the Analyst QS1.1 mascot.dll data import filter with default settings. The MS/MS data file generated was analysed using the Mascot 2.1 search engine (Matrix Science, London, UK) against UniProt April 2009 (7966092 sequences) or NCBInr March 2010 (10530540 sequenc- es) databases (Taxonomy, Mus musculus). The data was searched with tolerances of 0.2 Da for the precursor and fragment ions, trypsin as the cleavage enzyme, one missed cleavage, carbamido- methyl modification of cysteines as a fixed modification and methionine oxidation selected as a variable modification. More- over, the Mascot hits were considered valid with a protein score .50, except for known members of the PRC1 complex. Peptides identified in control IgG lane and specific IP lane were discarded. All proteins were checked against the UniProtKB database (uniprot.org). IgG heavy and light chains were omitted from the analysis, and therefore proteins migrating with a similar molecular size could not be identified.
pv. syringae B728a and characterize the ECF mutant phenotypes, especially in relation to survival as a plant-associated bacterium. Based on an investigation by Staron´ et al. , who classified ECF sigma factors into over 40 groups based on their modular architecture, the ECF sigma factors of strain B728a were identified by ECF group and named according to the report’s guidelines. Because iron availability exerts a strong influence on the expression of several virulence associated factors in P. syringae pv. syringae B728a [15,28], we characterized the roles played by three members of the FecI-type of ECF sigma factors in shaping the B728a host-pathogen interaction. We demonstrate that expression of the FecI-like ECF sigma factors is significantly up-regulated in conditions of iron stress and down-regulated in high iron conditions. In addition, ample quantities of iron are required for the production by B728a of a cyclic lipopeptide phytotoxin, syringomycin, which contributes to virulence . Because earlier studies of sigma factor mutants (i.e. rpoS, acsS, pvdS, and hrpL) in P. syringae pv. syringae did not reveal a regulatory effect on expression of the phytotoxin genes [17,30], a significant goal was to resolve whether the remaining uncharacterized ECF sigma factors included syringomycin production as a regulatory target. In addition to the three FecI-like ECF sigma factor genes, two ECF sigma factors placed in groups ECF11 and ECF18  were characterized for their contributions to pathogen resistance to specific environmental stresses. Transcriptional profiling by qRT- PCRanalysisof ECF sigma factor mutants measured the expression ofgenes encoding their associated anti-sigma and outer membrane receptor proteins, as well as the expression ofgenes associated, for example, with production of extracellular polysaccharides, fimbriae, and glycine betaine. Ultimately, each ECF sigma factor mutant was evaluated for growth and pathogenicity in leaves of a susceptible bean host.
Ten genes were randomly selected for confirmation using qRT-PCR to verify the accuracy of the identification of the DEG in the chip data. The 10 genes were signifi- cantly up-regulated in high royal jelly producing bees via qRT-PCRanalysis (Figure 4), which was consistent with chip data expression profiling analysis. This observation indicated the reliability of our chip expression profiling analysis. In NCBI, these 10 genes were annotated as fol- lows: dopamine receptor type D2 (Dop2) (GI: 20336614); Amt-2-like protein (GI: 67043607); similar to CG8862-PA (LOC551715) (GI: 110755554); clone hex71 hexamerin (hex71) (GI: 149939402); hypothetical protein LOC726515 (LOC726515) (GI: 110759535); similar to le- thal (1) G0168 CG33206-PA, isoform A (LOC411348) (GI: 110750767); similar to SHC-adaptor protein CG3715-PA (LOC412172) (GI: 66520065); similar to CG1998-PA, transcript variant 1 (LOC409360) (GI: 110749006); similar to LDLa domain containing chitin binding protein 1 CG8756-PD, isoform D, transcript vari- ant 1 (LOC551323) (GI: 110760992) and similar to multidrug resistance-associated protein 5 (LOC413947) (GI: 66538119). As they were significantly up-regulated in high royal jelly producing bees, these genes could play an important role in royal jelly production of Apis mellifera.
To evaluate the validity of Illumina analysis and assess the expression profiles in terms of specific mRNA abundances, 8 putative genes were selected and detected by qRT-PCR, and to further investigate the expression profiles of these genes, qRT- PCRof flowering tissues at different flowering developmental stages were performed separately using leaves of no-flowering plants as the control. Reverse transcription reactions were performed using 2 mg of RNA by M-MLVRT (Promega, USA) according to the manufacturer’s instructions. Sequences of 8 selected genes were obtained from the Moso bamboo genome database (http://www.ncgr.ac.cn/bamboo). Primers, picked by using the Primer 3 software (http://www.genome.wi. mit.edu/cgi- bin/primer/primer3.cgi), as shown in Additional file 13. Tono- plast intrinsic protein (TIP41), cited from Fan et al. , were used as the internal housekeeping gene control. Real-time PCR reactions were carried out with LightCycler480 System (Roche, USA) using SYBR Premix EX Taq kit (Roche, USA).The 20 m l reaction mixture contained 0.4 m l (10 m M) of each primer and 2 m l (50 ng) cDNA and 10 m l SYBR Green I Master according to the manufacturer’s instructions. Amplification reactions were per- formed as the following: 95 uC for 10 s, 60uC for 10 s, and 72uC for 20 s. All reactions were performed in triplicate, both technical and biological. Data was analyzed using Roche manager software.
stability . Ten generally used housekeeping primers for reference genes were designed for peanut and analyzed by GeNorm and NormFinder programs. Alcohol dehydrogen- ase (ADH3) showed to be the most stably expressed gene across samples, followed by 60S ribosomal protein L7 (60S) and yellow leaf specific 8 (YLS8) . However, to date, no endogenous controlgenes have been identified for other Arachis species, including the wild relatives which constitute a source of resistances to biotic and environmental constraints. In the present work, a simpli- fied qRT-PCR protocol based on SYBR reagent was used for the identification ofgenes with minimal expression variation in four Arachis species (A. magna; A. duranensis; A. stenosperma and A. hypogaea) subjected to biotic (Meloidogyne arenaria, Cercosporidium personatum) and abiotic (drought) stresses in roots and leaves. For that, we used our ESTs databank of wild Arachis  to survey for potential internal controlgenes and three distinct pro- grams (GeNorm, NormFinder, and BestKeeper) for their evaluation. Our data show that the combined use of these new internal controlgenes for normalization of target gene expression in qRT-PCR improves the accuracy and reliability of the analysisof gene expression in different species of the genus Arachis under different stresses.
Quantification of gene expression modulation is usually carried out by means of quantita- tive real-time reverse transcription-coupled PCR (qRT-PCR), and amplification data are nor- malized to one or more reference genes to account for intra- and inter-experimental variability. A reference gene needs to be expressed in the tissue considered, and ought not to be modulated by the physiological or pathological variables studied. Examples of good reference human genes include; ACTB and GAPDH in prostate cancer ; GAPDH and YWHAZ in idiopathic pregnancy-derived placenta ; and HPRT1 and SDHA in sepsis . As may be noticed from the above examples, these reference genes are housekeeping genes. Not all housekeeping genes, however, are suitable as reference genes as exemplified by the analysisof 81 ribosomal protein (Rpl) genes in 22 different tissues . Although all Rpl genes were expressed in all tis- sues tested, and as such qualify as housekeeping genes, none could be used as a reference gene owing to high expression variability. Thus, and as may be surmised from the list of tissues/ref- erence genes above, a gene or set ofgenes may serve as a reference in one tissue but may be inadequate for another. It may be inferred from these studies, therefore, that reference genes and genesets need to be determined for each tissue and in each physiological or pathological setting. This is particularly important in inflammatory conditions whereby the expression of many housekeeping genes can be modulated [46, 48, 49].
In order to further confirm and extend the results obtained from proteomic and transcriptomic analysis, we performed quantitative real-time PCR (qRT-PCR) analysis on 12 genes, all of which are very crucial in S metabolism pathways, including S uptake (Sulfate transporter1;2 gene, SULTR1;2), reduction (ATP sulfurylase gene, APS; APS reductase gene, APR) as well as on the genes related to S- containing amino acids synthesis (O-acetylserine(thiol)lyase gene, OASA; Cysteine synthase gene, OASB; Glutathione synthetase gene, GSH2) and other S derivates synthesis metabolisms (Glutathione S-transferase gene, GST3; Glutathione peroxidase gene, GPX6; Cytosolic thioredoxin gene, TRX5; Myrosinase gene, TGG2; S-adenosylmethionine synthetase gene, MTO3; S-adeno- sylmethionine decarboxylase gene, SAMDC). qRT-PCRanalysis showed that transcript expression level ofgenes related to primary sulfur assimilation, such as APR, APS1, OASA1 GSH2 and GST3, were up-regulated (Figure 3). However, the synthesis genesof some S-containing amino acids and derivatives (MTO3 and SAMDC) were down-regulated (Figure 3). The results were highly correlated with those of the array data, thus confirming the results from proteomic and transcriptomic studies. However, the change Figure 1. Protein expressions of Arabidopsis thaliana leaves after simulated acid rain (AR) treatment for 3 days (A). Molecular weight (Mr) in kilodaltons and pI of proteins are indicated on the left and top of the representative gel, respectively. Sixteen spots related to sulfur metabolism with at least a 2-fold change under AR stress are indicated. Close-up view of some differentially expressed protein spots (B).
Little is known about the types of organizational factors that directly contribute to aeronautical acci- dents. In contrast, there is a growing number of studies in the literature about the role that the error of the flight crew plays in the etiology of accidents. Studies suggest that up to 80% of all injuries are caused by unsafe pilot’s actions (Dismukes et al., 1999). This discrepancy on understanding the organizational factors is not surprising, given the fact that the pilot actions are more easily linked to the occurrence of an accident, while organizational factors are most of the times, temporally very distant from the event, making it difficult to connect them to an accident during an investigation (Wiegmann and Shappell, 2001). Some authors have argued that despite a growing awareness of organizational factors, they are often overlooked or are not identified by aeronautical accidents investigators (Heinrich et al., 1980; Yacavone, 1993; Maurino et al., 1995).
dexamethasone and TGF-b1. Thus, TGF-b3 appears to be superior to TGF-b1 (25). Our decision to use TGF-b3 was also based on a previous study from our group in which TGF-b3 was more efficient for inducing differentiation of MSCs derived from UCB into chondrocytes (9) than other growth factors (TGF-b3 was combined with dexamethasone to induce chondrogenesis). As a result, we were able to successfully induce the expression of type II collagen, aggrecan and SOX-9. In this study, chondrogenesis was induced in HAF-derived MSCs stimulated with TGF-b3 over 21 days using the micro- mass system. We confirmed this differentiation by analyzing the expression of the most important genes for the formation of articular cartilage (SOX-9, collagen type II and aggrecan) and confirmed the production of collagen type II at the protein level using histology or western blotting. Compared with normal human cartilage (used as a standard), cells under the micromass system showed significantly higher expression of the SOX-9 gene, as depicted in Figure 3A. This result indicates that it is possible to reproduce human chondrogen- esis in vitro, as SOX-9 is a key factor in inducing this process. Our results are reinforced by another study (5) in which the authors detected SOX-9 expression in cells under the micro- mass system but did not find a significant difference compared with that in cells under the pellet system, thereby indicating that transcription factor SOX-9 is essential for inducing chondrogenesis in both high-density systems.
Since IL-33 is exclusively present in the nucleus, the mechanism by which poly-IC induces IL-33 and enhances the expression of myelin is not clear. IL-33 is recognized as an “alarmin”, serving as a host response to pathogens and the release of IL-33 is caused by either cell death or necrosis. One possible mechanism by which IL-33 can be released in the absence of cell death is by activation of purinergic signaling pathways . Although ATP is a well-known source of energy, it also functions as a signaling molecule acting through specific purinergic receptors. In the CNS, astrocytes and oligodendrocytes express the adenosine receptor . Addition of adenosine to in vitro culture of astrocytes, stimulated to express IL-33, leads to release of IL-33 and its presence can be measured in culture supernatants . It is therefore conceivable that in an inflammatory milieu, the presence of adenosine could lead to release of IL-33 localized in the astrocytes, into the extracellular space, wherein it could bind and signal through its natural ligand, IL-33 receptor on oligodendrocytes and induce myelin gene expression (ST2/ IL1RAcP) [33, 39, 40]. IL-33 released in the extracellular space could also influence the polarization of macrophages to the M2 phenotype and enhance the recruitment of OPC and promote myeli- nation (Fig 15).
Comparando a expressão diferencial na pele entre bovinos resistentes e susceptíveis ao carrapato antes da infestação, foi possível observar a diferença de expressão de uma série de genes. Dentre os genes mais expressos nos animais susceptíveis, podem ser destacados os genes codificantes de: indoleamina 2,3- deoxigenase 1, de CXCL10 e da proteina quinase associada a cadeia zeta (ZAP70). A expressão da enzima indoleamina 2,3 deoxigenase 1 é induzida por INF- , sendo produzida principalmente por macrófagos e células dendríticas ativados. Quando expressa por essas células, age inibindo a proliferação de linfócitos T através da degradação de triptofano (HWU et al., 2000; MUNN et al., 2002). A quimiocina CXCL10 (também chamada IP-10) também tem sua expressão induzida por INF- , e a ela têm sido atribuída diversas funções tais como quioatração de monócitos, macrófagos, linfócitos T, células natural killer, promoção de adesão das células T às células endoteliais (DUFOUR et al., 2002). A proteína ZAP70 é naturalmente expressa células T e nk (natural killer), e está associada com o processo intracelular de ativação das células T (SKOV et al., 1997). A maior expressão destes genes nos animais susceptíveis antes da infestação artificial indica que possivelmente ainda havia nesses animais expressão de genes relacionados com resposta imune contra antígenos presentes na saliva do carrapato, provenientes de infestações naturais anteriores a realização do experimento .
To further examine whether the predicted target genes are directly regulated by miR-199a-5p, 3’ UTR segments of the corresponding genes containing the miR-199a-5p recognition region mutated region not recognizable by miR-199a-5p were sub-cloned downstream of the lucifer- ase reporter in psiCHECK-2 vectors (Applied Biosystems, Grand Island, NY, USA). The mu- tated sequences in the 3’ UTR segments of JunB and TGFBR1 were CATTACC and ACTAAC, respectively. The hsa-mir-199a-5p mimics and negative control (NC) mimics were purchased from Genepharma (Shanghai, China). 293T cells that were transiently transfected with hsa- mir-199a-5p or NC mimics at 15 or 50 nM of final concentration were co-transfected with Renilla luciferase reporter vector (195 ng/well) and Firefly luciferase reporter vector (5 ng/well) using Lipofectamine 2000 (Invitrogen) in 24-well plates. Cells were harvested at 48 hours post transfection and luciferase activities were analyzed by psiCHECK Dual Luciferase Reporter Assay (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Transfec- tion of miRNA or NC mimics into 786-O cells was carried out using Lipofectamine 2000 (Invi- trogen) according to manufacturer’s guide. Cells were collected for RNA and protein
Various age groups of C. gariepinus were obtained from our laboratory aquaculture facility. Catfish breeding, rearing and sample collection procedures were described earlier [38,44]. In brief, catfish were kept in glass water tanks supplied with filtered water and maintained under ambient photothermal conditions. They were fed tubeworms, ad libitum till they became fingerlings (5–6 mm). They were then provided minced goat liver or pelleted fish food till adulthood (around 400 dph). The catfish hatchlings were sacrificed at different time points i.e., 0 (less than 24 h post hatch), 5, 10, 20, 30, 50, 75, 100, 200 and 300 dph, and adult by briefly immobilizing them in mild ice-cold water dissolved with MS222 (Sigma, St. Louis, MO, USA). Brain was dissected out using fine scissors and forceps under stereo zoom microscope (Leica, Germany) except at day 0 wherein whole body was used. Since it was not possible to isolate brain from 10 and 20 dph larvae, we used entire head for total RNA preparation. All catfish experiments and sacrifice procedures were done by following the general animal ethical guidelines of Institutional Animal Ethical Committee (IAEC), however approval is not required for edible fish sacrifice. Five biological samples (n = 5) obtained from pooled brain tissues of 3-4 larvae for each sample were used for each time point of ontogeny study. To perform seasonal cycle studies, adult Figure 5. Catfish brain FTZ-F1 expression during gonadal ontogeny by qRT-PCR. Quantitative analysisof brain FTZ-F1 expression relative to b-actin expression during gonadal ontogeny of male and female catfish is reported as 2 2DCT . Values are mean 6 SEM, n = 5. *indicates the
The purpose of this study is to provide an independent recommendation of suitable refer- ence genes for normalisation in qRT-PCR analyses of narrow-leafed lupin. Here, we have eval- uated seven housekeeping genes previously trialed as candidate reference genes for the model legume, Medicago truncatula, by Kakar et al. . In that study, Kakar et al. compared 13 potential reference genes for stable expression across six organ types at up to three stages of maturity in vernalised M. truncatula plants. Our analysis incorporated a preliminary testing of the candidate reference genes for primer specificity, PCR amplification efficiency, and primer ability to discriminate between genomic DNA (gDNA) and cDNA of narrow-leafed lupin. The three most promising candidates were then evaluated using the NormFinder  and RefFin- der  algorithms for stable gene expression with a greater number of factors, including: firstly, testing for consistent expression in two lines of narrow-leafed lupin (a representative wild and representative domestic line); secondly, stability within and across seven organ types; thirdly, stability of expression over time (via collection of organs at three developmental stages); and lastly, consistent expression with and without vernalisation (i.e., a prolonged period of exposure to cold winter temperatures, which enables floral competency in warm spring conditions ). Based on these results, we present our recommendations for suitable reference genes for qRT-PCR studies of narrow-leafed lupin.
A metodologia do Luminex consiste basicamente na técnica de reação em cadeia da polimerase (PCR) associada à hibridização com microesferas de poliestireno acopladas a sondas específicas para cada alvo. As referencias para o protocolo de ciclagem, iniciados e sondas foram específicos para cada painel de patógenos e regiões alvos estão a seguir: vírus (Rotavírus, NSP3; Norovírus, ORF1-ORF2; Astrovírus, Capsid; Sapovírus, RdRp-capsid; Adenovírus, Hexon) (Liu J et al., 2011); E. coli enteropatogênicas; E. coli enteroagregativa, aaiC e aatA; E.coli entropatogênica, bfpA e eaeA; E.coli enterotoxigênica, LT e ST; e E. coli enterohemorragica, stx1, stx2 e eaeA) (Taniuchi et al., 2013); outras bactérias (Shigella spp, ipaH; Salmonella spp, invA; Campylobacter spp., cadF; Aeromonas spp, aerolysin e Vibrio spp, t toxR) (Liu J et al., 2012); protozoários (Giárdia spp, 18S rRNA; Cryptosporidium spp, COWP e Entamoeba histolytica, 18S rRNA) (Taniuchi et al., 2011). A detecção foi realizada por meio do equipamento Luminex Bio-Plex 200 System (Bio-Rad). A identificação por Luminex foi utilizada como diagnóstico inicial para realizar testes subsequentes.
Stevens (1946, p. 677) defines measurement as “the assignment of numerals to objects or events according to rules”. There are four scales of measurement that are quite different and cannot be used interchangeably even though they may be represented by numerals. The scales are divided into metric (quantitative) and non-metric (qualitative) scales. The quantitative measures are interval variables, with discrete levels in which the interval between each category is equal and well defined (e.g. number of people, number of computers); and ratio variables, with no data restriction and allowance of any value, even fractions (e.g. income and height). The qualitative measures are: nominal variables, in which designated numerals simply represent categories without implying in amounts of an attribute or characteristic and categorization ofdata does not associate a hierarchy level (e.g. gender); and ordinal variables, whose concept is broader and will be presented next.
Tremendous amount of efforts have been focused on discover- ing predictive and prognostic biomarkers for gastric cancer . Due to the disease complicity of gastric cancer, it still remains a major challenge to identify clinical useful biomarkers. With the recognition of the broad impact of miRNAs on multi targets and pathways, it is of our interest to identify a new class of predictive and prognostic biomarkers in gastric cancer based on miRNAs. There are several advantages of using miRNAs as biomarker compared to mRNA as miRNAs have broad regulatory function, relatively small numbers and better stability in archival FFPE samples . We report here that expression levels of miR-181b and miR-21 constitute strong prognostic factors in advanced gastric cancer patients on both S-1/Oxaliplatin and Doxiflur- idine/Oxaliplatin regimens. The Clinical and pathologic param- eters of gastric cancer patients treated with Doxifluridine/ Oxaliplatin or S-1/Oxaliplatin were listed in Table 1.