DDRT PCR PDF

Wasko b. Differential Display DD is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored.

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Wasko b. Differential Display DD is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored.

Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs Differentially Expressed Transcripts , using Leporinus macrocephalus , a large commercially exploited South American species, as a fish design.

The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR Variable Number of Tandem Repeats core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides.

The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach. In the last years, diverse genetic studies have focused on the analysis of differentially expressed genes DETs - Differentially Expressed Transcripts or, in other words, genes expressed as messenger RNAs mRNA which differ in abundance among cell types or specific tissues, and which may be regulated by chemical, physiological, and environmental mechanisms Liang and Pardee, These studies allow gene characterization and evaluation of transcript expression levels in relation to extrinsic factors.

Therefore, it is possible to compare samples isolated at different developing stages, collected from different environments, or from different tissues or genders Cervigni et al. A large number of methods have been used to isolate and characterize DETs, and DD Differential Display Liang and Pardee, represents one of the most popular techniques concerning this approach. The DD methodology has been widely used in differential expression studies since it shows high efficiency and is characterized for its simplicity, reduced cost and for not requiring any previous knowledge about the target genome Liang and Pardee, Moreover, the differential display can simultaneously visualize increased or decreased expression of numerous mRNAs from many samples, and requires relatively small amounts of starting material Pardee and McClelland, For that reason, modifications like the use of tailored primers to amplify members of a particular gene family Jurecic et al.

Despite polymerase chain reaction PCR -based mRNA differential display has been widely used for identifying differentially expressed transcripts in a variety of species e. Liang, , some animals, as fishes, generally show a few data related to this approach. The majority of differential gene expression studies in fish species, through the use of DD, are restricted to North American and European species, or to commercial fishes, as Salmoniformes, which claim to generate important results to fish farming Parrington and Coward, In this way, studies related to South American species are poorly exploited.

Besides, works describing conditions to improve the results of fish DD are still required in this animal group. Dakis and Kouretas , for example, have described some modifications in the DD technique applied to fish samples, which were characterized using a combination of longer arbitrary primers 25 and mer together with mer anchored primer for PCR reactions, and the use of a nondenaturing polyacrylamide gel to analyze PCR products. Thereby, the present work describes some different modifications of the DD technique in order to improve the results of this method in relation to DETs isolation in fish species.

The improved approach permitted the isolation of differentially expressed transcripts in adult males and females of this species. As there is no genomic data for L. Total RNA samples obtained from each male and female were incubated with DNase I to remove DNA contamination, which represents a further source of false-positive bands that can be observed on differential display gels.

After DNase treatment, small aliquots of each sample were loaded onto an agarose gel to check for RNA integrity. Alves-Costa and Wasko, ; Alves-Costa et al. Preliminary analyses were performed to determine primers that provide superior and reproducible amplification results.

As so, cDNA amplification was achieved using decamer oligonucleotides commonly used in RAPD analyses and also oligonucleotides that were designed based in VNTR core sequences that correspond to minisatellite short and highly conserved regions Jeffreys et al.

The use of the mer primers, that commonly generate multiple bands for different organisms Lee et al. Moreover, the amplification reactions usually resulted on several faint and diffuse bands, and the results were generally not reproducible Figure 1.

These faint bands often lead to a misinterpretation of the data. In addition, just a reduced number of candidate differentially expressed transcripts could be identified when comparing males and females of L. A single amplified fragment that may correspond to a differentially expressed transcript is highlighted box.

Some studies evidenced that primers with 13 bases or longer have better effects in the DD efficiency Zhao et al. These data are consistent with the proposal that DDRT-PCR reactions need special adjustments for each species samples since its fingerprinting patterns may vary from one cell type to another Blaes et al. The single amplified fragments that may correspond to differentially expressed transcripts are highlighted boxes.

Despite this optimization in the DD approach in brain tissues of a fish species, our results led to the identification of a restricted number of amplification products, even using longer primers. The DD methodology generally leads to the visualization of 5 to amplification products in polyacrylamide gel McClelland et al. The unworkable achievement of a complex amplification pattern with a high number of bands can be due to the high PCR stringency conditions, through an annealing temperature of 55 o C, or to the use of RNA sample pools that can avoid the detection of individual variations.

Despite the relative reduced number of amplified fragments around 5 to 15 bands visualized on polyacrylamide gels, some of them could be detected only in males or females, which indicate that they correspond to presumptive differentially expressed transcripts between the two samples.

Thirty one cDNAs appeared to be differentially expressed on males and females of L. Therefore, the improved DD approach using VNTR core sequences as primers in RT-PCR led to the identification of putative sex differentially expressed transcripts in this fish species and may be used as a strategy in future studies related to this approach in order to obtain consistent data. Alves-Costa, FA. Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

Genetica, vol. Anais da Academia Brasileira de Ciencias, vol. Blaes, N. Differential display fingerprints: new approach to characterize smooth muscle cells and human coronary atherectomy tissues. Pathologie Biologie, vol. Cervigni, GDL. Gene expression in diplosporous and sexual Eragrostis curvula genotypes with differing ploidy levels.

Plant Molecular Biology, vol. Dakis, D. In Vivo Athens, Greece , vol. Dhorne-Pollet, S. Validation of novel reference genes for RT-qPCR studies of gene expression in Xenopus tropicalis during embryonic and post-embryonic development. Developmental dynamics : an official publication of the American Association of Anatomists, vol. Elis, S. Search for the genes involved in oocyte maturation and early embryo development in the hen. BMC Genomics, vol.

Graf, D. Nucleic Acids Research, vol. Huang, YJ. Progress in Biochemistry and Biophysics, vol. Jeffreys, AJ. Nature, vol.

Jurecic, R. Differential mRNA display using anchored oligo-dT and long sequence-specific primers as arbitrary primers. Trends in genetics: TIG, vol. Kavar, T. Identification of genes involved in the response of leaves of Phaseolus vulgaris to drought stress.

Molecular Breeding, vol. Kim, TW. Identification of replicative senescence-associated genes in human umbilical vein endothelial cells by an annealing control primer system.

Experimental Gerontology, vol. Kim, YS. Identification of differentially expressed genes using an annealing control primer system in stage III serous ovarian carcinoma. BMC Cancer, vol. Kumar, RR. Characterization of differentially expressed stress-associated proteins in starch granule development under heat stress in wheat Triticum aestivum L. Lee, KF. A comparative study of gene expression in murine embryos developed in vivo, cultured in vitro, and cocultured with human oviductal cells using messenger ribonucleic acid differential display.

Biology of Reproduction, vol. Liang, P. Current progress in differential display methods and applications. Methods: A Companion to Methods in Enzymology. New York: Academic Press. A decade of differential display. BioTechniques, vol. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.

Science, vol. Recent advances in differential display. Current Opinion in Immunology, vol. Ma, J. Single-primer PCR correction: a strategy for false-positive exclusion.

Genetics and molecular research: GMR, vol. McClelland, M. Malhotra, K.

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Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles

Metrics details. Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Both methods have been used merely as qualitative gene discovery tools so far. We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: i cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, ii intensity of bands is determined by densitometry, iii densitometric values are normalized, and iv intensity ratio is calculated for each pair of corresponding bands.

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Differential display

The host-fungus interaction is characterized by changes in gene expression in both host and pathogen. Several limitations and drawbacks to this procedure have now been addressed, including the large number of false-positive results and the difficulty in confirming differential expression. Modifications that simplify the reaction time, allow the use of minute quantities of RNA, or address unusual species- or gene-specific sequences have been reported. DDRT-PCR has been used to address biological questions in mammalian systems, including cell differentiation, cell activation, cell stress, and identification of drug targets. In microbial pathogenesis and plant pathogenesis, DDRT-PCR has allowed the identification of virulence factors, genes involved in cell death, and signaling genes. In Histoplasma capsulatum and C. Although very few reports have been published in medical mycology, studies in mammalian, nonfungal microbial, and plant pathogen systems are easily applied to basic questions in fungal pathogenesis and antifungal therapeutics.

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Differential-Display Reverse Transcription-PCR (DDRT-PCR)

Protocol DOI: The invention of polymerase chain reaction PCR and its application to amplification of reverse transcribed cDNA copies of mRNA has opened new possibilities for the development of techniques for identification of changes in gene expression patterns. In contrast to the standard RT-PCR technique, where individual mRNAs are detected in a qualitative and sometimes also quantitative manner using gene-specific primers, in these new techniques a set of short primers with arbitrary sequences is used to generate amplified cDNA fragments from a large number of mRNAs used before for DNA fingerprinting 3. Basically, the identification of every individual mRNA species expressed should be possible provided a sufficiently large number of primer pairs is used 4. Therefore, we give only a very brief description of the principle.

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