mRNA-display
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Cíl: Včasná diagnostika karcinomu prostaty (CaP) je zcela zásadní pro záchyt karcinomu omezeného jen na žlázu s možností následné kurabilní léčby. Prostatický specifický antigen (PSA) je velice dobrý prostředek pro včasnou diagnostiku, ale jeho nevýhodou je nízká pozitivní prediktivní hodnota, jejíž následkem je velké množství zbytečných biopsií. Z tohoto důvodu je potřeba vývoje nových specifičtějších detekčních testů. Jedním ze slibných testů je DD3PCA3, což je gen kódující pro prostatickou tkáň specifickou mRNA, která je výrazně exprimována ve tkáni CaP. Cílem prezentované práce je vyhodnocení diagnostického potenciálu DD3PCA3 v detekci CaP stanovením exprese mRNA tohoto genu ve tkáni prostaty u mužů s karcinomem prostaty a benigní hyperplazií prostaty. Materiál a metoda: Celkem bylo vyšetřeno 186 pacientů. Ve skupině pacientů s podezřením na karcinom prostaty jsme v rámci biopsie prostaty odebrali vždy jeden vzorek na expresi DD3PCA3. Na základě histologického vyšetření byli pacienti rozděleni do skupiny s benigní hyperplazií prostaty, celkem 100 pacientů a 86 pacientů s prokázaným karcinomem prostaty (z tohoto počtu byla u 12 pacientů stanovena diagnóza prostatické intraepiteliální neoplazie (PIN)). Byla stanovena celková mRNA a kvantifikována celková exprese DD3PCA3 a PSA s užitím Q RT PCR metodiky. Poměr PCA3/PSA mRNA byl stanoven u obou skupin pacientů. Výsledky: V rámci předložené studie bylo prokázáno, že hladiny exprese mRNA genu DD3PCA3 (Ct) (hodnoty Ct cyklu), ve kterém dojde ke křížení s přímkou nejnižšího prahu detekce tzv. thresholdu. Čím je hodnota nižší, tím vyšší je množství kopií) ve tkáni prostaty byly signifikantně vyšší (p < 0,045) u pacientů s CaP ve srovnání s pacienty s BPH. Nebyly prokázány signifikantní rozdíly v expresi mRNA pro gen DD3PCA3 (Ct) mezi skupinami pacientů v závislosti na pokročilosti onemocnění (lokální vs. lokálně pokročilý a metastatický CaP) a v závislosti na Gleasonově skóre (dobře až středně diferencovaný CaP vs. špatně diferencovaný karcinom). Závěr: Specificita detekce mRNA DD3PCA3 ve tkáni je perspektivní metodikou pro detekci CaP a rozlišení pacientů s CaP a BPH.
Aim: Early diagnosis of prostate cancer (PCa) in organ confined stage with following radical treatment are only potential currative approach in PCa. Prostatic specific antigen (PSA) is very helpful in early diagnosis, but the main disadvantage is a low positive predictive value, which results in a high number of uselless biopsies. For that reason we need new tests with better parameters. One promising is DD3PCA3, which is a prostate-specific non-coding mRNA that is highly over-expressed in prostate tumor cells. The aim of study was to evaluate diagnostic potential of DD3PCA3 for PCa diagnostic. Material and methods: We examined altogether 186 patients. In group of patients with suspicion of PCa we collected one tissue specimen core for PCA3 espression in tissue. According to the histologically verification 100 patients with benign prostatic hyperplasia (BPH), and 86 patients with prostate cancer (12 patients from them had prostatic intraepithelial neoplasia (PIN)). Total RNA were isolated, PCA3 and PSA expression quantified using Q RT PCR method. The PCA3/PSA m RNA ratio distribution was determined for both subject groups. Results: It was found that levels of mRNA expressions of DD3PCA3 (Ct) were significantly higher (p < 0.045) in patients with prostate cancer than in patients with benign prostatic hyperplasia. We found no statistically diferencies in patients with prostate cancer in levels of mRNA expressions of DD3PCA3 (Ct) between patients with organe confined and advanced (metastatic) dissease and according to Gleason score. Conclusion: The specificity of mRNA DD3PCA3 detection seems to be perspective for early differential diagnosis between patients with BPH and patients with prostate cancer.
- MeSH
- antigeny nádorové genetika MeSH
- DNA primery diagnostické užití MeSH
- financování organizované MeSH
- hyperplazie prostaty diagnóza genetika MeSH
- lidé MeSH
- messenger RNA diagnostické užití MeSH
- nádory prostaty MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- sekvence nukleotidů genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
SCOPE: CYP3A4 is the most important drug-metabolizing enzyme regulated via the vitamin D receptor (VDR) in the intestine. However, less is known about VDR in the regulation of CYP3A4 and other drug-metabolizing enzymes in the liver. METHODS AND RESULTS: This study investigates whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) regulates major cytochrome P450 enzymes, selected phase I and II enzymes, and transporters involved in xenobiotic and steroidal endobiotic metabolism in 2D and 3D cultures of human hepatocytes. The authors found that 1α,25(OH)2 D3 increases hepatic CYP3A4 expression and midazolam 1'-hydroxylation activity in 2D hepatocytes. The results are confirmed in 3D spheroids, where 1α,25(OH)2 D3 has comparable effect on CYP3A4 mRNA expression as 1α-hydroxyvitamin D3 , an active vitamin D metabolite. Other regulated genes such as CYP1A2, AKR1C4, SLC10A1, and SLCO4A1 display only mild changes in mRNA levels after 1α,25(OH)2 D3 treatment in 2D hepatocytes. Expression of other cytochrome P450, phase I and phase II enzyme, or transporter genes are not significantly influenced by 1α,25(OH)2 D3 . Additionally, the effect of VDR activation on CYP3A4 mRNA expression is abolished by natural dietary compound sulforaphane, a common suppressor of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). CONCLUSION: This study proposes that VDR or vitamin D supplementation is unlikely to significantly influence liver detoxification enzymes apart from CYP3A4.
- MeSH
- cytochrom P-450 CYP3A * genetika MeSH
- hepatocyty MeSH
- lidé MeSH
- messenger RNA MeSH
- receptory kalcitriolu genetika MeSH
- stanovení celkové genové exprese MeSH
- systém (enzymů) cytochromů P-450 genetika MeSH
- vitamin D farmakologie MeSH
- xenobiotika * farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). RESULTS: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. CONCLUSIONS: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.
- MeSH
- lidé MeSH
- messenger RNA analýza krev metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí přístrojové vybavení metody MeSH
- senzitivita a specificita MeSH
- stanovení celkové genové exprese přístrojové vybavení metody MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Limited accessibility to intestinal epithelial tissue in wild animals and humans makes it challenging to study patterns of intestinal gene regulation, and hence to monitor physiological status and health in field conditions. To explore solutions to this limitation, we have used a noninvasive approach via fecal RNA-seq, for the quantification of gene expression markers in gastrointestinal cells of free-range primates and a forager human population. Thus, a combination of poly(A) mRNA enrichment and rRNA depletion methods was used in tandem with RNA-seq to quantify and compare gastrointestinal gene expression patterns in fecal samples of wild Gorilla gorilla gorilla (n = 9) and BaAka hunter-gatherers (n = 10) from The Dzanga Sangha Protected Areas, Central African Republic. RESULTS: Although only a small fraction (< 4.9%) of intestinal mRNA signals was recovered, the data was sufficient to detect significant functional differences between gorillas and humans, at the gene and pathway levels. These intestinal gene expression differences were specifically associated with metabolic and immune functions. Additionally, non-host RNA-seq reads were used to gain preliminary insights on the subjects' dietary habits, intestinal microbiomes, and infection prevalence, via identification of fungi, nematode, arthropod and plant RNA. CONCLUSIONS: Overall, the results suggest that fecal RNA-seq, targeting gastrointestinal epithelial cells can be used to evaluate primate intestinal physiology and gut gene regulation, in samples obtained in challenging conditions in situ. The approach used herein may be useful to obtain information on primate intestinal health, while revealing preliminary insights into foraging ecology, microbiome, and diet.
- MeSH
- feces * MeSH
- gastrointestinální trakt metabolismus MeSH
- Gorilla gorilla genetika MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- poly A genetika MeSH
- sekvenování transkriptomu * MeSH
- stanovení celkové genové exprese * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Hazelnut (Corylus), which has high commercial and nutritional benefits, is an important tree for producing nuts and nut oil consumed as ingredient especially in chocolate. While Corylus avellana L. (Euro-pean hazelnut, Betulaceae) and Corylus colurna L. (Turkish hazelnut, Betulaceae) are the two common hazelnut species in Europe, C. avellana L. (Tombul hazelnut) is grown as the most widespread hazelnut species in Turkey, and C. colurna L., which is the most important genetic resource for hazelnut breeding, exists naturally in Anatolia. We generated the transcriptome data of these two Corylus species and used these data for gene discovery and gene expression profiling. Total RNA from young leaves, flowers (male and female), buds, and husk shoots of C. avellana and C. colurna were used for two different libraries and were sequenced using Illumina HiSeq4000 with 100 bp paired-end reads. The transcriptome data 10.48 and 10.30 Gb of C. avellana and C. colurna, respectively, were assembled into 70,265 and 88,343 unigenes, respectively. These unigenes were functionally annotated using the TRAPID platform. We identified 25,312 and 27,051 simple sequen-ce repeats (SSRs) for C. avellana and C. colurna, respectively. TL1, GMPM1, N, 2MMP, At1g29670, CHIB1 unigenes were selected for validation with qPCR. The first de novo transcriptome data of C. co-lurna were used to compare data of C. avellana of commercial importance. These data constitute a valuable extension of the publicly available transcriptomic resource aimed at breeding, medicinal, and industrial research studies.
- MeSH
- líska * genetika metabolismus MeSH
- ořechy MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Turecko MeSH
- MeSH
- bariatrická chirurgie MeSH
- gastrektomie * využití MeSH
- hmotnostní úbytek fyziologie MeSH
- lidé MeSH
- mediátory zánětu * metabolismus škodlivé účinky MeSH
- messenger RNA genetika MeSH
- monocyty sekrece MeSH
- obezita metabolismus MeSH
- prospektivní studie MeSH
- stanovení celkové genové exprese MeSH
- tuková tkáň * cytologie metabolismus sekrece MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- grafy a diagramy MeSH
- práce podpořená grantem MeSH
Teeth have been a focus of interest for many centuries--due to medical problems with them. They are the hardest part of the human body and are composed of three mineralized parts--enamel, dentin and cementum, together with the soft pulp. However, saliva also has a significant impact on tooth quality. Proteomic research of human teeth is now accelerating, and it includes all parts of the tooth. Some methodological problems still need to be overcome in this research field--mainly connected with calcified tissues. This review will provide an overview of the current state of research with focus on the individual parts of the tooth and pellicle layer as well as saliva. These proteomic results can help not only stomatology in terms of early diagnosis, identifying risk factors, and systematic control.
RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3' to 5' on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.
- MeSH
- editace RNA * MeSH
- genom mitochondriální genetika MeSH
- genom protozoální genetika MeSH
- izoformy RNA genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- RNA mitochondriální genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- sestřih RNA MeSH
- stanovení celkové genové exprese metody MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- Trypanosomatina genetika metabolismus MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.
- MeSH
- lidé MeSH
- messenger RNA krev chemie genetika izolace a purifikace MeSH
- odběr vzorku krve MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- stabilita RNA MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
