|
| Status |
Public on Mar 25, 2010 |
| Title |
No pregnancy vs calf delivery rep 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Biopsied embryos consisting of 60-70% of inner cell mass and trophectoderm
|
| Organism |
Bos taurus |
| Characteristics |
embryo biopsy result: no pregnancy
stage of embryo: blastocyst stage embryo
|
| Growth protocol |
Embryos were developed in vio and flushed afer 7 days of development
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from three independent pools consisting of 10 of embryo biopsies that resulted in calf delivery or no pregnancy using the PicoPureTM RNA isolation kit according to the manufacturer's instruction. Genomic DNA contamination was removed by performing on column DNA digestion using RNase-free DNase set (Qiagen, Hilden, Germany).
|
| Label |
Cy3
|
| Label protocol |
3 µg of amplified RNA from each embryo biopsies ( calf delivery and No pregnancy) was used as template in reverse transcription reactions incorporating amino-modified dUTPs into the cDNA using the cyscribe post-labelling kit (Amersham Biosciences, Freiburg, Germany). The Purified aminoallyl labelled cDNA samples from each group were differentially labelled indirectly using N-hydroxysuccinate-derived Cy3 and Cy5 dyes a. To avoid variation due to dye coupling, amplified RNA samples from the same group were labelled reversibly either with Cy3 or Cy5 for target and dye-swap hybridizations. The reaction was then purified with CyScribeTM GFXTM Purification kit (Amersham Biosciences, Freiburg, Germany)
|
| |
|
| Channel 2 |
| Source name |
Biopsied embryos consisting of 60-70% of inner cell mass and trophectoderm
|
| Organism |
Bos taurus |
| Characteristics |
stage of embryo: blastocyst stage embryo
embryo biopsy result: calf delivery
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from three independent pools consisting of 10 of embryo biopsies that resulted in calf delivery or no pregnancy using the PicoPureTM RNA isolation kit according to the manufacturer's instruction. Genomic DNA contamination was removed by performing on column DNA digestion using RNase-free DNase set (Qiagen, Hilden, Germany).
|
| Label |
Cy5
|
| Label protocol |
3 µg of amplified RNA from each embryo biopsies ( calf delivery and No pregnancy) was used as template in reverse transcription reactions incorporating amino-modified dUTPs into the cDNA using the cyscribe post-labelling kit (Amersham Biosciences, Freiburg, Germany). The Purified aminoallyl labelled cDNA samples from each group were differentially labelled indirectly using N-hydroxysuccinate-derived Cy3 and Cy5 dyes a. To avoid variation due to dye coupling, amplified RNA samples from the same group were labelled reversibly either with Cy3 or Cy5 for target and dye-swap hybridizations. The reaction was then purified with CyScribeTM GFXTM Purification kit (Amersham Biosciences, Freiburg, Germany)
|
| |
|
| |
| Hybridization protocol |
Labelled cDNA hybridised using the ready made bovine cDNA array (BlueChip version 3, kindly provided by prof. Andre Sirard, (Laval university, Quebec, Canada).The array consisting of more than 2200 targets genes derived from suppression subtractive hybridization of bovine embryo and tissues
|
| Scan protocol |
Array scanning and image analysis was performed using Axon GenePix 4000B scanner and GenePix Pro analysis software (version 4.0) (Axon Instruments, Foster City, CA), respectively
|
| Description |
Analysis was performed by comparing the gene expression of embryo biopsies that resulted in calf delivery and embryos biopsies resulted in no pregnancy
|
| Data processing |
Data analysis was perfomed using R programme and bioconductor packages. The values from each channel were log2 transformed and normalized using the LOESS algorithm to remove intensity dependent effects within the calculated values. a mean log2-transformed value of (Cy5/Cy3) was calculated for the replicates to obtain one value per clone. Differentially expressed genes were obtained using linear models for microarray data (LIMMA)
|
| |
|
| Submission date |
Mar 24, 2010 |
| Last update date |
Mar 24, 2010 |
| Contact name |
Dessie Salilew-Wondim |
| E-mail(s) |
dsalilew@uni-bonn.de
|
| Organization name |
University of Bonn
|
| Department |
Animal breeding and husbandry group
|
| Street address |
Endenicher Allee 15
|
| City |
Bonn |
| State/province |
NRW |
| ZIP/Postal code |
D-53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10249 |
| Series (2) |
| GSE21047 |
Bovine pre-transfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer (embryo study) |
| GSE21049 |
Bovine pre-transfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer |
|
