Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture

2009 ◽  
Vol 76 (1) ◽  
pp. 38-47 ◽  
Author(s):  
Sadie L. Smith ◽  
Robin E. Everts ◽  
Li-Ying Sung ◽  
Fuliang Du ◽  
Raymond L. Page ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
In Vitro Maturation ◽  
Bovine Embryos

scholarly journals Gene expression profiling of human oocytes following in vivo or in vitro maturation

2008 ◽  
Vol 23 (5) ◽  
pp. 1138-1144 ◽  
Author(s):  
Gayle M. Jones ◽  
David S. Cram ◽  
Bi Song ◽  
M. Cristina Magli ◽  
Luca Gianaroli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
In Vitro Maturation ◽  
Human Oocytes

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals In Vitro Transcription Amplification and Labeling Methods Contribute to the Variability of Gene Expression Profiling with DNA Microarrays

2006 ◽  
Vol 8 (2) ◽  
pp. 183-192 ◽  
Author(s):  
Changqing Ma ◽  
Maureen Lyons-Weiler ◽  
Wenjing Liang ◽  
William LaFramboise ◽  
John R. Gilbertson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Dna Microarrays ◽  
In Vitro Transcription

Intraplatform Reproducibility and Technical Precision of Gene Expression Profiling in Four Laboratories Investigating 112 Leukemia Samples: The DACH Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2383-2383
Author(s):  
Alexander Kohlmann ◽  
Elisabeth Haschke-Becher ◽  
Barbara Wimmer ◽  
Ariana Huber-Wechselberger ◽  
Sandrine Meyer-Monard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Control Group ◽  
Unsupervised Hierarchical Cluster ◽  
Daily Routine ◽  
Total Rna

Abstract Gene expression profiling has the potential to offer consistent objective diagnostic test results once a standardized protocol is established. We investigated the robustness, precision, and reproducibility of this technology and present data that complements the Microarray Innovations in LEukemia study (MILE study). In four laboratories, located in Germany (D), Austria (A), and Switzerland (CH) (DACH study), replicates of 112 patient samples were analyzed using the AmpliChip Leukemia research test. Patient samples were centrally collected and diagnosed in daily routine at the Munich Leukemia Laboratory and represented 8 distinct classes of acute and chronic leukemias, with non-leukemia as control group. After purification of the mononuclear cells by Ficoll density centrifugation, 4 × 5 million cells were frozen in lysis buffer and stored at −80°C. Equipped with identical instruments, software, and reagents, study operators were trained on the microarray sample preparation protocol using total RNA from commercially available cell lines. Upon receipt of the frozen lysates each of the four laboratories purified the total RNA from the 112 technical quadruplicates. 99.3% (445/448) of the sample preparations were successfully performed. On average, 8.4 μg, 7.2 μg, 7.4 μg, or 7.5 μg of total RNA, respectively, were isolated from the mononuclear cells from the four laboratories. In three samples less than 1.0 μg of total RNA was obtained and thus the preparation failed. Bland-Altman plots of agreement showed that any two centers were unlikely to have more than an 8.3 μg difference in yield of total RNA from the same sample. On average there was between 0.1 μg to 1.2 μg difference in total RNA yield from the same sample. Further processing of the 445 samples resulted in 437 (98.2%) successfully performed in vitro transcription reactions, i.e. obtained cRNA yield of >8.0 μg. On average there was between 0.4 μg to 7.4 μg difference in cRNA yield from the same sample. After hybridization to microarrays on average, 46.1%, 48.6%, 46.5%, and 47.3% of probe sets were detected as present with mean scaling factors of 4.3, 2.9, 3.9, and 3.7, respectively. The mean values and standard deviations of distributions of the coefficient of variation (CV) within each site over all the probe sets of the quantile normalized signals on the chip were 27.2% (StdDev: 12.3%), 27.0% (StdDev: 12.3%), 27.3% (StdDev: 12.3%), 26.9% (StdDev: 12.4%), respectively. Furthermore, in unsupervised hierarchical cluster and principal component analyses replicates from the same patient always clustered closely together, with no indications of association between gene expression profiles due to different operators or laboratories. In conclusion, we demonstrated that microarray analysis can be performed with remarkably high inter-laboratory reproducibility and with comparable quality and high technical precision across laboratories.

scholarly journals Gene expression profiling of acute type A aortic dissection combined with in vitro assessment†

2017 ◽  
Vol 52 (4) ◽  
pp. 810-817 ◽  
Author(s):  
Naoyuki Kimura ◽  
Kyoko Futamura ◽  
Mamoru Arakawa ◽  
Naoko Okada ◽  
Fabian Emrich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Aortic Dissection ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Type A ◽  
Acute Type ◽  
Type A Aortic Dissection ◽  
In Vitro Assessment
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