Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

Background: MicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings. Results: We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification. Conclusions: MiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification

Deciphering the genetic architecture of prolificacy related traits in an experimental Iberian x Meishan F2 intercross

Els caràcters reproductius són de gran interès en la indústria porcina per tal de millorar l'eficiència productiva. En estudis previs, utilitzant un creuament experimental F2 entre les races Ibèric (Ib) i Meishan (Me) es van identificar varis QTL afectant varis caràcters reproductius (Noguera et al., 2009, Fernandez-Rodriguez et al., 2010, Rodriguez et al., 2005). En particular, es van identificar 2 QTL amb efecte al nombre de garrins nascuts vius (NV) i al nombre total de garrins nascuts (NT) localitzats en els cromosomes porcins 13 (SSC13) i SSC17 (Noguera et al., 2009). Per tal de poder identificar els gens responsables dels QTL en el SSC13, es van analitzar quatre gens candidats (ITIH1, ITIH3, ITIH4 and MUC4). La caracterització del clúster de gens ITIH va permetre identificar que aquests tenen un efecte sobre el NV però que és independent dels QTL associats a la mida de la ventrada. Els anàlisis del gen MUC4, que es troba localitzat dins l'interval de confiança del QTL en el SSC13, van determinar una associació significativa entre un SNP dins d'aquest gen i els caràcters NV i NT, tot i que l'efecte era superior pel NV. A més, l'expressió del gen MUC4 en l'úter és dues vegades superior en truges d'alta prolificitat. Per tal de millorar el nostre coneixement envers l'arquitectura genètica dels caràcters relacionats amb la prolificitat, es va analitzar el transcriptoma a nivell d'expressió gènica en úter així com també els nivells d'expressió de miRNAs tant en úter com en ovari. Aquests estudis es van realitzar utilitzant truges F2 IbxMe que presentaven fenotips extrems pels nivells de prolificitat definits com el nombre d'embrions (NE) units a l'úter a dia 30-32 de la gestació. En l'úter de les truges d'alta prolificitat es van identificar 101 gens (upregulated) implicats en la resposta inflamatòria enfront als estímuls i en el desenvolupament del teixit muscular. Per altra banda, 196 gens (downregulated) es van relacionar amb el desenvolupament del teixit muscular, l'organització d'unió cel·lular, en processos d'adhesió, en la regulació biològica, en processos del sistema muscular i circulatori i en el transport. L'estudi del microRNAoma va identificar els miR-125b-5p, miR-200C-3p, miR-23b-3p, miR-23-3p i miR-99-5p com els més abundants en l'úter de les truges gestants, mentre que l'expressió de miR-139- 5p, miR-150-5p, miR-27-3p i miR-20-5P es van associar amb els nivells de prolificitat. Entre tots els possibles gens diana per als miRNAs relacionats amb la prolificitat, es troben 32 gens localitzats dins l'interval de confiança per als QTL de prolificitat i, per tant, es van proposar com a bons gens candidats per a ser estudiats. Entre aquests, es troba el gen MUC4 que és de gran interès per a ser el responsable del QTL en el SSC13 ja que reuneix varis criteris; es localitza dins l'interval de confiança del QTL en el SSC13, té un efecte sobre la mida de la ventrada, el seu nivell d'expressió es relaciona amb els nivells de prolificitat i pot ser regulat pel miR-150-5p, que també es va trobar diferencialment expressat en relació amb els nivells de prolificitat. D'altra banda, en ovari, els miR-146a-5p i miR-142-3p, involucrats en processos del sistema immunològic i en la homeòstasis cel·lular, estan diferencialment expressats en relació amb els nivells de prolificitat. Quatre gens diana per aquests miRNAs (LRRK1, CCL8, CPEB2 and BAT1) es troben dins l'interval de confiança per als QTL amb efecte per la prolificitat, fet que fa que siguin bons candidats a ser estudiats. Finalment, es va dissenyar un nou mètode RT-qPCR molt específic, sensible i precís per tal de mesurar els nivell d'expressió dels miRNAs mitjançant l'ús d'encebadors d'ADN.Reproductive traits are of great interest in the swine industry to improve the pig efficiency production. In a previous study, several QTL affecting reproductive traits were identified in an Iberian (Ib) x Meishan (Me) F2 population (Noguera et al., 2009, Fernandez-Rodriguez et al., 2010, Rodriguez et al., 2005). In particular, two QTL for the number of piglets born alive (NBA) and the total number of piglets born (TNB) were located on porcine chromosomes 13 (SSC13) and SSC17 (Noguera et al., 2009). To identify genes responsible for the prolificacy QTL on SSC13, four candidate genes (ITIH1, ITIH3, ITIH4 and MUC4) were analysed. Analyses for ITIH gene cluster showed that these genes had an effect on NBA independent of litter size QTL. Results for MUC4 gene, located within the confidence interval of the QTL on SSC13, determined that a SNP within the MUC4 gene was associated with NBA and TNB although the effect was stronger for NBA. In addition, uterine MUC4 expression was two-fold higher in high prolificacy sows. To better understand the genetic basis of prolificacy related traits, transcriptome analyses, which included the study of uterine gene expression as well as the miRNA expression profile in the uterus and in the ovary, was performed. For this, IbxMe F2 sows displaying extreme phenotypes regarding the prolificacy levels defined as the number of embryos (NE) attached to the uterus at day 30-32 of the gestation were used. In uterus of high prolificacy sows, 101 genes involved in the inflammatory response to stimulus and muscle tissue development were upregulated whereas 196 genes that participated in muscle tissue development, cell junction organization and adhesion, biological regulation, muscle and circulatory system processes, and transport were downregulated. The microRNAome identified the miR-125b-5p, miR-200c-3p, miR-23b-3p, miR-23a-3p and miR-99a-5p as the most abundant miRNAs in uterus of pregnant sows while expression of miR-139-5p, miR-150-5p, miR-27a-3p and miR-20-5p was associated with prolificacy levels. Among predicted gene target for uterine prolificacy-related miRNAs, 32 were located within the confidence interval of the QTL for NBA and TNB and therefore, they are proposed to be good candidate genes to be further investigated. Importantly, among these candidate genes, it is found MUC4 gene which is of great interest to be the responsible for the prolificacy QTL on SSC13 because it fulfilled several criteria: it is located within the QTL confidence interval with an effect on NBA and TNB, its expression level varies regarding the prolificacy level of sows, and it is targeted by miR-150-5p, a miRNA that was also found diferentially expressed regarding the prolificacy levels. On the other hand, ovarian miR-146a-5p and miR-142-3p, involved in immune system processes and cellular homeostasis, were differentially expressed regarding prolificacy levels. Four predicted target genes (LRRK1, CCL8, CPEB2 and BAT1), located within confidence intervals for prolificacy QTL, are good candidate genes to be studied for QTL on litter size. Alternatively, we have designed a new RT-qPCR methodology, by using DNA primers to measure miRNA expression, which is highly specific, sensitive and accurate

Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy

Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages.This study was funded by the Spanish Ministerio de Ciencia e Innovación (grant AGL2004-08368-C03/GAN). RN Pena received a contractual grant from INIA. M Martínez-Giner received a predoctoral fellowship from MICINN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Analysis of porcine MUC4 gene as a candidate gene for prolificacy QTL on SSC13 in an Iberian × Meishan F2 population

Background: Reproductive traits, such as prolificacy, are of great interest to the pig industry. Better understanding of their genetic architecture should help to increase the efficiency of pig productivity through the implementation of marker assisted selection (MAS) programmes. Results: The Mucin 4 (MUC4) gene has been evaluated as a candidate gene for a prolificacy QTL described in an Iberian × Meishan (Ib × Me) F2 intercross. For association analyses, two previously described SNPs (DQ124298:g.243A>G and DQ124298:g.344A>G) were genotyped in 347 pigs from the Ib × Me population. QTL for the number of piglets born alive (NBA) and for the total number of piglets born (TNB) were confirmed on SSC13 at positions 44 cM and 51 cM, respectively. The MUC4 gene was successfully located within the confidence intervals of both QTL. Only DQ124298:g.344A>G MUC4 polymorphism was significantly associated with both NBA and TNB (P-value < 0.05) with favourable effects coming from the Meishan origin. MUC4 expression level was determined in F2 sows displaying extreme phenotypes for the number of embryos (NE) at 30-32 days of gestation. Differences in the uterine expression of MUC4 were found between high (NE ≥ 13) and low (NE ≤ 11) prolificacy sows. Overall, MUC4 expression in high prolificacy sows was almost two-fold increased compared with low prolificacy sows. Conclusions: Our data suggest that MUC4 could play an important role in the establishment of an optimal uterine environment that would increase embryonic survival during pig gestation

miRNA Expression Profile Analysis in Kidney of Different Porcine Breeds

MicroRNAs (miRNAs) are important post-transcriptional regulators in eukaryotes that target mRNAs repressing their expression. The uncertain process of pig domestication, with different origin focuses, and the selection process that commercial breeds suffered, have generated a wide spectrum of breeds with clear genetic and phenotypic variability. The aim of this work was to define the miRNAs expression profile in kidney of several porcine breeds. Small RNA libraries from kidney were elaborated and high-throughput sequenced with the 454 Genome Sequencer FLX (Roche). Pigs used were classified into three groups: the European origin group (Iberian breed and European Wild Boar ancestor), European commercial breeds (Landrace, Large White and Piétrain breeds) and breeds with Asian origin (Meishan and Vietnamese breeds). A total of 229 miRNAs were described in the pig kidney miRNA profile, including 110 miRNAs out of the 257 previously described pig miRNAs and 119 orthologous miRNAs. The most expressed miRNAs in pig kidney microRNAome were Hsa-miR-200b-3p, Ssc-miR-125b and Ssc-miR-23b. Moreover, 5 novel porcine miRNAs and 3 orthologous miRNAs could be validated through RT-qPCR. miRNA sequence variation was determined in 116 miRNAs, evidencing the presence of isomiRs. 125 miRNAs were differentially expressed between breed groups. The identification of breed-specific miRNAs, which could be potentially associated to certain phenotypes, is becoming a new tool for the study of the genetic variability underlying complex traits and furthermore, it adds a new layer of complexity to the interesting process of pig evolution

Endometrial gene expression profile of pregnant sows with extreme phenotypes for reproductive efficiency

Prolificacy can directly impact porcine profitability, but large genetic variation and low heritability have been found regarding litter size among porcine breeds. To identify key differences in gene expression associated to swine reproductive efficiency, we performed a transcriptome analysis of sows' endometrium from an Iberian x Meishan F2 population at day 30-32 of gestation, classified according to their estimated breeding value (EBV) as high (H, EBV > 0) and low (L, EBV < 0) prolificacy phenotypes. For each sample, mRNA and small RNA libraries were RNA-sequenced, identifying 141 genes and 10 miRNAs differentially expressed between H and L groups. We selected four miRNAs based on their role in reproduction, and five genes displaying the highest differences and a positive mapping into known reproductive QTLs for RT-qPCR validation on the whole extreme population. Significant differences were validated for genes: PTGS2 (p = 0.03; H/L ratio = 3.50), PTHLH (p = 0.03; H/L ratio = 3.69), MMP8 (p = 0.01; H/L ratio = 4.41) and SCNN1G (p = 0.04; H/L ratio = 3.42). Although selected miRNAs showed similar expression levels between H and L groups, significant correlation was found between the expression level of ssc-miR-133a (p < 0.01) and ssc-miR-92a (p < 0.01) and validated genes. These results provide a better understanding of the genetic architecture of prolificacy-related traits and embryo implantation failure in pigs

Functional Implications of Human-Specific Changes in Great Ape microRNAs

microRNAs are crucial post-transcriptional regulators of gene expression involved in a wide range of biological processes. Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra-and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs (miR-299-3p, miR-503-3p, miR508-3p and miR-541-3p) revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species.Peer reviewe

The role of viral and host microRNAs in the Aujeszky's disease virus during the infection process

Porcine production is a primary market in the world economy. Controlling swine diseases in the farm is essential in order to achieve the sector necessities. Aujeszky's disease is a viral condition affecting pigs and is endemic in many countries of the world, causing important economic losses in the swine industry. microRNAs (miRNAs) are non-coding RNAs which modulates gene expression in animals, plants and viruses. With the aim of understanding miRNA roles during the Aujeszky's disease virus [ADV] (also known as suid herpesvirus type 1 [SuHV-1]) infection, the expression profiles of host and viral miRNAs were determined through deep sequencing in SuHV-1 infected porcine cell line (PK-15) and in an animal experimental SuHV-1 infection with virulent (NIA-3) and attenuated (Begonia) strains. In the in vivo approach miR-206, miR-133a, miR-133b and miR-378 presented differential expression between virus strains infection. In the in vitro approach, most miRNAs were down-regulated in infected groups. miR-92a and miR-92b-3p were up-regulated in Begonia infected samples. Functional analysis of all this over expressed miRNAs during the infection revealed their association in pathways related to viral infection processes and immune response. Furthermore, 8 viral miRNAs were detected by stem loop RT-qPCR in both in vitro and in vivo approaches, presenting a gene regulatory network affecting 59 viral genes. Most described viral miRNAs were related to Large Latency Transcript (LLT) and to viral transcription activators EP0 and IE180, and also to regulatory genes regarding their important roles in the host-pathogen interaction during viral infection

Natural Course of the Diffusing Capacity of the Lungs for Carbon Monoxide in COPD: Importance of Sex

[Background] The value of the single-breath diffusing capacity of the lungs for carbon monoxide (Dlco) relates to outcomes for patients with COPD. However, little is known about the natural course of Dlco over time, intersubject variability, and factors that may influence Dlco progression.[Research Question] What is the natural course of Dlco in patients with COPD over time, and which other factors, including sex differences, could influence this progression?[Study Design and Methods] We phenotyped 602 smokers (women, 33%), of whom 506 (84%) had COPD and 96 (16%) had no airflow limitation. Lung function, including Dlco, was monitored annually over 5 years. A random coefficients model was used to evaluate Dlco changes over time.[Results] The mean (± SE) yearly decline in Dlco % in patients with COPD was 1.34% ± 0.015%/y. This was steeper compared with non-COPD control subjects (0.04% ± 0.032%/y; P = .004). Sixteen percent of the patients with COPD, vs 4.3% of the control subjects, had a statistically significant Dlco % slope annual decline (4.14%/y). At baseline, women with COPD had lower Dlco values (11.37% ± 2.27%; P < .001) in spite of a higher FEV1 % than men. Compared with men, women with COPD had a steeper Dlco annual decline of 0.89% ± 0.42%/y (P = .039).[Interpretation] Patients with COPD have an accelerated decline in Dlco compared with smokers without the disease. However, the decline is slow, and a testing interval of 3 to 4 years may be clinically informative. The lower and more rapid decline in Dlco values in women, compared with men, suggests a differential impact of sex in gas exchange function.[Trial Registry] ClinicalTrials.gov; No.: NCT01122758; URL: www.clinicaltrials.govThis study was funded in part by an unrestricted grant from AstraZeneca, and also by the COPD Research Program of the Spanish Respiratory Society (PII de EPOC of SEPAR).Peer reviewe

New GOLD classification: longitudinal data on group assignment

Rationale: Little is known about the longitudinal changes associated with using the 2013 update of the multidimensional GOLD strategy for chronic obstructive pulmonary disease (COPD). Objective: To determine the COPD patient distribution of the new GOLD proposal and evaluate how this classification changes over one year compared with the previous GOLD staging based on spirometry only. Methods: We analyzed data from the CHAIN study, a multicenter observational Spanish cohort of COPD patients who are monitored annually. Categories were defined according to the proposed GOLD: FEV1%, mMRC dyspnea, COPD Assessment Test (CAT), Clinical COPD Questionnaire (CCQ), and exacerbations-hospitalizations. One-year follow-up information was available for all variables except CCQ data. Results: At baseline, 828 stable COPD patients were evaluated. On the basis of mMRC dyspnea versus CAT, the patients were distributed as follows: 38.2% vs. 27.2% in group A, 17.6% vs. 28.3% in group B, 15.8% vs. 12.9% in group C, and 28.4% vs. 31.6% in group D. Information was available for 526 patients at one year: 64.2% of patients remained in the same group but groups C and D show different degrees of variability. The annual progression by group was mainly associated with one-year changes in CAT scores (RR, 1.138; 95%CI: 1.074-1.206) and BODE index values (RR, 2.012; 95%CI: 1.487-2.722). Conclusions: In the new GOLD grading classification, the type of tool used to determine the level of symptoms can substantially alter the group assignment. A change in category after one year was associated with longitudinal changes in the CAT and BODE index