User:Z3414515: Difference between revisions
(→Lab 1) |
(→Lab 1) |
||
| Line 37: | Line 37: | ||
FVB/N mice were kept for 12 hours under light/dark cycle and were fed regularly. A Pregnant Mare’s Serum (5 IU) was injected into a 4-7 weeks old female. After 44 hours a human chorionic gonadotropin (5 IU) injection was given. The females then mated with the FVB/N strain studs (males). 19-21 hours later the females were sacrificed and the oviducts were collected. Oocytes were collected. The embryos were then cultured in KSOM medium. | FVB/N mice were kept for 12 hours under light/dark cycle and were fed regularly. A Pregnant Mare’s Serum (5 IU) was injected into a 4-7 weeks old female. After 44 hours a human chorionic gonadotropin (5 IU) injection was given. The females then mated with the FVB/N strain studs (males). 19-21 hours later the females were sacrificed and the oviducts were collected. Oocytes were collected. The embryos were then cultured in KSOM medium. | ||
Gene expression analysis | |||
Extraction of RNA from mouse unfertilised oocytes using Arcturus PicoPure RNA isolation kit was done. Agilent Bioanalyser was used to measure the RNA quality and concentration. One embryo yielded 128 pg of total RNA on average. For each final protocol, three biological replicas of all the stages were collected. | |||
TaqMan Array Cards analysis | |||
RQ Manager version 1.2.2 (Applied Biosystems) were used to analyse Ct values. Hprt1 and Psmb6 were the endogenous controls which were used for normalisation. | |||
Expression analysis from public sequencing dataset | |||
Gene Expression Omnibus database was used to obtain the normalised RPKM values for human and mouse pre-implantation stages. The p-values were calculated for the pairs i.e. oocytes and 4-cell blastomeres and etc. The p-values below 0.05 were significant. In human and mouse, the average values for each stage between embryos in the same biological stages were calculated. | |||
'''Result summary''' | |||
Analysis of two independent human pre-implantation microarray datasets were done in order to define the genes with consistent gene expression profiles between embryo stages. The probes which had significant changes in both datasets were considered for further analysis. Probes in the “Up-down” cluster were up regulated whereas the probes in “Down” cluster were down regulated. Genes were selected from each cluster “Up”, Up-down” and “Down” for analysis of expression profile of mouse pre-implantation embryo by qPCR. A gene was included if its ortholog was found in any of the following samples in MGI: oocyte, unfertilized oocyte, fertilized oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, 16-cell embryo, blastocyst. In the mouse, 55 genes with orthologs were selected for gene expression profiling. Also expression patterns of the selected genes in the mouse were studied. The maternal gene expression profile was seen to be shared in more than half of the mouse orthologs for genes “Up” and “Up-down” clusters. All the PRAME and most SSX, MAGEA and GAGE family members in human microarray were of “up-down” cluster. However, in the pre-implantation human embryo, the selected families’ genes had dynamic expression profiles. | |||
Revision as of 00:11, 8 August 2014
Lab Attendance
Lab 1 --Z3414515 (talk) 12:46, 6 August 2014 (EST)
Practice
http://www.ncbi.nlm.nih.gov/pubmed
<pubmed>4118885</pubmed>
My Type in a Group
Teamworker
A Teamworker is the oil between the cogs that keeps the machine that is the team running smoothly. They are good listeners and diplomats, talented at smoothing over conflicts and helping parties understand one other without becoming confrontational. Since the role can be a low-profile one, the beneficial effect of a Teamworker can go unnoticed and unappreciated until they are absent, when the team begins to argue, and small but important things cease to happen. Because of an unwillingness to take sides, a Teamworker may not be able to take decisive action when it is needed.
Lecture Reviews
Lecture 1
Course introduction for embryology as well as the history of embryologists and how the diagrams of embryo changed through time as more advance technology was available. Guidelines to the course was mentioned as well as the assessments and type of work expected for this course.
Lecture 2
In the fertilization lecture the most interesting concept for me was the polar bodies and the sry gene. Every other concepts such as gametes, mitosis, meiosis and fertilization was familiar. Polar bodies and the sry gene was a completely new idea for me. Meiosis 1 releases first polar body and meiosis 2 releases the second polar body. Sometimes meiosis 1 releases first and third polar bodies.
Individual Assessments
Lab 1
Reference: Pmid25089626
<pubmed>25089626</pubmed>
Method summary:
Microarray Analysis
Raw data on Affymetrix GeneChip HGU133 were obtained from the ArrayExpress for human preimplantation embryos. The invariant set normalisation method was used and via using the Li-Wong method, the expression values were extracted from PM-values. The arrays were normalised independently and Li-Wong method was applied to all normalised arrays to get a summary of the expression measurements. Using Bayesian approach, differential expression between the consecutive development stages was analysed.
Embryo Collection
FVB/N mice were kept for 12 hours under light/dark cycle and were fed regularly. A Pregnant Mare’s Serum (5 IU) was injected into a 4-7 weeks old female. After 44 hours a human chorionic gonadotropin (5 IU) injection was given. The females then mated with the FVB/N strain studs (males). 19-21 hours later the females were sacrificed and the oviducts were collected. Oocytes were collected. The embryos were then cultured in KSOM medium.
Gene expression analysis
Extraction of RNA from mouse unfertilised oocytes using Arcturus PicoPure RNA isolation kit was done. Agilent Bioanalyser was used to measure the RNA quality and concentration. One embryo yielded 128 pg of total RNA on average. For each final protocol, three biological replicas of all the stages were collected.
TaqMan Array Cards analysis
RQ Manager version 1.2.2 (Applied Biosystems) were used to analyse Ct values. Hprt1 and Psmb6 were the endogenous controls which were used for normalisation.
Expression analysis from public sequencing dataset
Gene Expression Omnibus database was used to obtain the normalised RPKM values for human and mouse pre-implantation stages. The p-values were calculated for the pairs i.e. oocytes and 4-cell blastomeres and etc. The p-values below 0.05 were significant. In human and mouse, the average values for each stage between embryos in the same biological stages were calculated.
Result summary
Analysis of two independent human pre-implantation microarray datasets were done in order to define the genes with consistent gene expression profiles between embryo stages. The probes which had significant changes in both datasets were considered for further analysis. Probes in the “Up-down” cluster were up regulated whereas the probes in “Down” cluster were down regulated. Genes were selected from each cluster “Up”, Up-down” and “Down” for analysis of expression profile of mouse pre-implantation embryo by qPCR. A gene was included if its ortholog was found in any of the following samples in MGI: oocyte, unfertilized oocyte, fertilized oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, 16-cell embryo, blastocyst. In the mouse, 55 genes with orthologs were selected for gene expression profiling. Also expression patterns of the selected genes in the mouse were studied. The maternal gene expression profile was seen to be shared in more than half of the mouse orthologs for genes “Up” and “Up-down” clusters. All the PRAME and most SSX, MAGEA and GAGE family members in human microarray were of “up-down” cluster. However, in the pre-implantation human embryo, the selected families’ genes had dynamic expression profiles.
