scholarly journals Evaluating the Performance of Oligonucleotide Microarrays for Bacterial Strains with Increasing Genetic Divergence from the Reference Strain

2010 ◽  
Vol 76 (9) ◽  
pp. 2980-2988 ◽  
Author(s):  
Seungdae Oh ◽  
Deborah R. Yoder-Himes ◽  
James Tiedje ◽  
Joonhong Park ◽  
Konstantinos T. Konstantinidis
Keyword(s):  
Reference Strain ◽  
Experimental Results ◽  
Hybridization Signal ◽  
Target Pair ◽  
Oligonucleotide Microarrays ◽  
Bacterial Strains ◽  
Gene Detection ◽  
False Negatives ◽  
Expression Studies ◽  
Gene Expression Studies

ABSTRACT DNA oligonucleotide microarrays (oligoarrays) are being developed continuously; however, several issues regarding the applicability of these arrays for whole-genome DNA-DNA strain comparisons (genomotyping) have not been investigated. For example, the extent of false negatives (i.e., no hybridization signal is observed when the amino acid sequence is conserved but the nucleotide sequence has diverged to a level that does not allow hybridization) remains speculative. To provide quantitative answers to such questions, we performed competitive DNA-DNA oligoarray (60-mer) hybridizations with several fully sequenced (tester) strains and a reference strain (whose genome was used to design the oligoarray probes) of the genus Burkholderia and compared the experimental results obtained to the results predicted based on bioinformatic modeling of the probe-target pair using the available sequences. Our comparisons revealed that the fraction of the total probes that provided experimental results consistent with the predicted results decreased substantially with increasing divergence of the tester strain from the reference strain. The fractions were 90.8%, 84.3%, and 77.4% for tester strains showing 96% 89%, and 80% genome-aggregate average nucleotide identity (ANI) to the reference strain, respectively. New approaches to determine gene presence or absence based on the hybridization signal, which outperformed previous approaches (e.g., 92.9% accuracy versus 86.0% accuracy) and to normalize across different array experiments are also described. Collectively, our results suggest that the performance of oligoarrays is acceptable for tester strains showing >90% ANI to the reference strain and provide useful guidelines for using oligoarray applications in environmental gene detection and gene expression studies with strains other than the reference strain.

scholarly journals Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kim Hoa Ho ◽  
Annarita Patrizi
Keyword(s):  
Gene Expression ◽  
Choroid Plexus ◽  
Sensory Deprivation ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Experimental Conditions ◽  
Brain Repair ◽  
Expression Studies ◽  
Testing Algorithms ◽  
Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

scholarly journals Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1272
Author(s):  
Judit Tajti ◽  
Magda Pál ◽  
Tibor Janda
Keyword(s):  
Gene Expression ◽  
Avena Sativa ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Nutritional Value ◽  
Tissue Type ◽  
Future Research ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

scholarly journals Isolation of Human Adrenocortical Aldosterone-Producing Cells by a Novel Immunomagnetic Beads Method

Endocrinology ◽  
2010 ◽  
Vol 151 (3) ◽  
pp. 1375-1380 ◽  
Author(s):  
Brasilina Caroccia ◽  
Ambrogio Fassina ◽  
Teresa Maria Seccia ◽  
Chiara Recarti ◽  
Lucia Petrelli ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Zona Glomerulosa ◽  
Multiple Features ◽  
Pheochromocytoma Cells ◽  
Expression Studies ◽  
Neural Cell Adhesion ◽  
Multiple Characteristics ◽  
Normal Human ◽  
Aldosterone Producing Adenoma ◽  
Gene Expression Studies

We detected intense CD56 immunostaining in the zona glomerulosa (ZG) and medulla of the normal human adrenal gland and therefore identified CD56, the neural cell adhesion molecule, as a membrane antigen specific for the ZG, aldosterone-producing adenoma (APA), and chromaffin cells. The APA and pheochromocytoma cells, which are histogenetically derived from the ZG and medulla, respectively, also showed intense CD56 immunostaining. Based on these findings we developed a strategy for isolating cells from the ZG and APA using CD56 immunobinding to magnetic beads. Morphology, gene expression studies, and aldosterone measurement confirmed that CD56 positive (+) cells were ZG and APA cells. Analysis of CD56+ cells under light and phase contrast microscopy evidenced that these cells formed clumps, as the ZG cells usually do; with electron microscopy they showed multiple features typical of a steroidogenic phenotype. Expression levels of the CD56 and the aldosterone synthase (CYP11B2) gene were markedly higher in CD56+ cells than CD56− cells (+1600 and +2100% increase, respectively). Moreover, aldosterone secretion was higher (+1380%) from CD56+ cells than from CD56− cells. Hence, this novel methodology allows isolation of a pure population of ZG and APA cells exhibiting multiple characteristics of the aldosterone-producing cells.

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