Highly efficient method for isolation of total RNA from adipose tissue

BACKGROUND: RNA extraction is a crucial step for monitoring gene expression. Poor RNA quality (including degradation and remaining impurities) can result in misleading results. Isolation of RNA from animal tissues with high lipid content can be challenging. Especially, it is not trivial to isolate high quality RNA with a reasonable yield from adipose tissue. The aim of this study was to provide an optimized protocol for isolating total RNA from adipose tissue. This was achieved by combining the advantages of the two routinely used methods, TRI Reagent® and miRNeasy. FINDINGS: The miRNeasy method results in cleaner samples but more prone to degradation while the TRI Reagent® method results in samples contaminated with salts and solvents but more intact. The new protocol combines the best of both methods resulting in RNA of high quality and suitable for downstream experiments like RT-qPCR, microarrays and high-throughput sequencing. CONCLUSIONS: The current protocol for total RNA isolation from adipose tissue yields sufficient amount of high quality total RNA free of contaminants

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

Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

<p>Abstract</p> <p>Background</p> <p>Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR.</p> <p>Results</p> <p>In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (<it>ACTB</it>), beta-2-microglobulin (<it>B2M</it>), glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), hydroxymethylbilane synthase (<it>HMBS</it>), hypoxanthine phosphoribosyltransferase 1 (<it>HPRT1</it>), ribosomal protein L4 (<it>RPL4</it>), succinate dehydrogenase complex subunit A (<it>SDHA</it>), TATA box binding protein (<it>TPB</it>)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (<it>YWHAZ</it>). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): <it>ACTB</it>/<it>RPL4</it>, <it>TBP</it>, <it>HPRT</it>, <it>HMBS</it>, <it>YWHAZ</it>, <it>SDHA</it>, <it>B2M </it>and <it>GAPDH</it>.</p> <p>Conclusion</p> <p>Expression stability varies greatly between genes. <it>ACTB, RPL4</it>, <it>TPB </it>and <it>HPRT1 </it>were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that <it>ACTB </it>and <it>RPL4 </it>are good reference genes for high abundant transcripts while <it>TPB </it>and <it>HPRT1 </it>are good reference genes for low abundant transcripts in expression studies across different pig tissues.</p

Discovery and annotation of novel microRNAs in the porcine genome by using a semi-supervised transductive learning approach

Despite the broad variety of available microRNA (miRNA) prediction tools, their application to the discovery and annotation of novel miRNA genes in domestic species is still limited. In this study we designed a comprehensive pipeline (eMIRNA) for miRNA identification in the yet poorly annotated porcine genome and demonstrated the usefulness of implementing a motif search positional refinement strategy for the accurate determination of precursor miRNA boundaries. The small RNA fraction from gluteus medius skeletal muscle of 48 Duroc gilts was sequenced and used for the prediction of novel miRNA loci. Additionally, we selected the human miRNA annotation for a homology-based search of porcine miRNAs with orthologous genes in the human genome. A total of 20 novel expressed miRNAs were identified in the porcine muscle transcriptome and 27 additional novel porcine miRNAs were also detected by homology-based search using the human miRNA annotation. The existence of three selected novel miRNAs (ssc-miR-483, ssc-miR484 and ssc-miR-200a) was further confirmed by reverse transcription quantitative real-time PCR analyses in the muscle and liver tissues of Göttingen minipigs. In summary, the eMIRNA pipeline presented in the current work allowed us to expand the catalogue of porcine miRNAs and showed better performance than other commonly used miRNA prediction approaches. More importantly, the flexibility of our pipeline makes possible its application in other yet poorly annotated non-model species.info:eu-repo/semantics/acceptedVersio

Identificação por "Multiplex PCR" do sorotipo monofásico e atípico Salmonella enterica subsp. enterica sorotipo 1,4,[5],12:i:-, no Estado de São Paulo, Brasil: freqüência e resistência antimicrobiana

Salmonella spp. are the etiologic agents of salmonellosis, a worldwide spread zoonoses causing foodborne outbreaks and clinical diseases. By serological identification, Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- accounted for 8.8% of human and 1.6% of nonhuman Salmonella strains isolated in São Paulo State, during 1991-2000. A total of 28.6% of them amplified a fragment corresponding to H:1,2 (flagellar phase two) through PCR analysis and were further assigned as S. Typhimurium. Antimicrobial resistance was detected in 36.3% of the 369 PCR-negative strains tested, including the multiresistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin.Salmonella spp. é o agente etiológico da salmonelose, zoonose mundialmente distribuída e responsável por surtos de doenças transmitidas por alimentos e doenças clínicas. Sorologicamente, Salmonella enterica subsp. enterica sorotipo 1,4,[5],12:i:- correspondeu a 8,8% e 1,6% das cepas de Salmonella de origem humana e não-humana, respectivamente, isoladas no Estado de São Paulo, no decênio 1991-2000. Aproximadamente 28,6% destas cepas amplificaram o fragmento correspondente a H:1,2 (fase flagelar dois) em testes de PCR e foram, então, identificadas como S. Typhimurium. Das 369 cepas negativas em PCR, 36,3% apresentou resistência antimicrobiana, incluindo multirresistência a ampicilina, cloranfenicol, sulfonamidas, tetraciclina e estreptomicina

EST analysis on pig mitochondria reveal novel expression differences between developmental and adult tissues

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The mitochondria are involved in many basic functions in cells of vertebrates, and can be considered the power generator of the cell. Though the mitochondria have been extensively studied there appear to be only few expression studies of mitochondrial genes involving a large number of tissues and developmental stages. Here, we conduct an analysis using the PigEST resource 1 which contains expression information from 35 tissues distributed on one normalized and 97 non-normalized cDNA libraries of which 24 are from developmental stages. The mitochondrial PigEST resource contains 41,499 mitochondrial sequences. Results The mitochondrial EST (Expressed Sequence Tag) sequences were assembled into contigs which covers more than 94 percent of the porcine mitochondrial genome, with an average of 976 EST sequences per nucleotide. This data was converted into expression values for the individual genes in each cDNA library revealing differential expression between genes expressed in cDNA libraries from developmental and adult stages. For the 13 protein coding genes (and several RNA genes), we find one set of six genes, containing all cytochrome oxidases, that are upregulated in developmental tissues, whereas the remaining set of seven genes, containing all ATPases, that are upregulated in adult muscle and brain tissues. Further, the COX I (Cytochrome oxidase subunit one) expression profile differs from that of the remaining genes, which could be explained by a tissue specific cleavage event or degradation pattern, and is especially pronounced in developmental tissues. Finally, as expected cDNA libraries from muscle tissues contain by far the largest amount (up to 20%) of expressed mitochondrial genes. Conclusion Our results present novel insight into differences in mitochondrial gene expression, emphasizing differences between adult and developmental tissues. Our work indicates that there are presently unknown mechanisms which work to customize mitochondrial processes to the specific needs of the cell, illustrated by the different patterns between adult and developmental tissues. Furthermore, our results also provide novel insight into how in-depth sequencing can provide significant information about expression patterns.Published versio