08/10/09 -
P.24 cells are growing well and are confluent, so were passaged from P.24 to P.25. P.33 cells are growing more slowly, which is usual for human fibroblast cells so new medium was added.
09/10/09 -
P.33 cells passaged to P.34
14/10/09 -
Met with Dylan Edwards (my supervisor) and Tracey Swingler, a postdoctoral research associate in Ian Clarke's research group who will be helping me with my project. Tracey is working on the identification and characterisation of microRNAs involved in chondrogenesis and osteoarthritis, so is experienced in studying microRNAs. I arranged to meet with Tracey on 16/10/09 to discuss my project.
16/10/09 -
The first part of showing a link between zmpste24 and miR-34 is to use bioinformatics to show a theoretical binding site for miR-34 on zmpste24. Tracey demonstrated how to use software such as TargetScan, Pictar, mirbase to find targets for miR-34 or to find all miRNA targets for a protein.
Passaged cells from P.25 to P.26 and passaged one flask of P.34 cells to P.35, but the other flask of P.34 cells had not grown as quickly so new medium was added.
17/10/09-19/10/09 -
Searched through bioinformatics databases. Zmpste24 did not show a binding site for miR-34 in either the well conserved or poorly conserved sites in any of the databases I checked (targetscan, pictar and microRNA.org). Targetscan brought up 114 miRNA targets for Zmpste24 and microRNA.org produced 3,022 theoretical gene targets for miR-34. This may be due to the fact that these databases are constantly being updated and it may have caused zmpste24 to have been lost from the list of gene targets for miR-34, especially if it is a poorly conserved site.
20/10/09 -
Met with Dylan and Tracey to discuss problem of not finding zmpste24 in the databases. When searching the databases lamin A/C came up as a gene target for miR-34, so it was decided to use the lamin A/C protein in the probes instead. Zmpste24 regulates lamin A/C so inhibition of lamin A/C has the same effect on the cells and will produce the same senescence phenotype. Tamas Dalmay, who works with microRNAs investigating their roles in various projects, suggested looking through the sequence of the 3'-UTR of Zmpste24, which can be found using the database Ensembl, to find the miR-34 seed sequence. It is unlikely I will be able to find this sequence as the binding site in Zmpste24 will not be perfectly complimentary, so I could be looking for a number of different combinations of the sequence with any of the bases included.
I looked through the databases again and found on microRNA.org a potential binding site for miR-34 on Zmpste24, starting at bp 1481. I also found the splice variant of lamin A/C which has the longest 3'-UTR using Ensembl. I can use this information and try to find the miR-34 seed sequence with in it.
Tracey and I have decided to start extracting RNA from my cells on 23/10/09, the RNA can be frozen until we are ready to use it. I will also be taking pictures of the cells I have grown which will allow us to visualise the difference between senescent and non-senescent cells.
21/10/09 -
I have had a brief look at the literature to try and establish which form or miR-34 (a, b or c, *,5p or 3p) is the most abundant or shows an effect. So far it seems miR-34a is the most abundant and commonly used form in experiments, but miR-34c is also used. More reading is needed to make a more informed decision.
Thursday 22 October 2009
Wednesday 7 October 2009
Inductions and beginning to grow tissue
29/09/2009
Today I had the building induction in the Biomedical Research Centre (BMRC) with Alba Warn. Alba took me through all health and safety procedures and took me around the lab showing me where each research group works, the communal areas, waste disposal, first aid kits and fire exits. Main points to note were:
Chief Technicians - Alba Warn & Jasmine Waters
Emergency Evacuation Procedures - Follow signs to Fire Exits
Alert others by striking 'break glass' boxes to set alarm
Assembly points: Across DMU loading bay for East Wing, and grass between BMRC and Sainsbury Centre for West Wing.
First aid information is on posters at each end of the lab
Labcoats to be worn at all times, along with gloves, goggles, face visor as appropriate (e.g. wear face visor and protective gloves when handling liquid nitrogen).
01/10/2009
Today was my induction to the tissue culture lab, again with Alba Warn. I was shown where all equipment for the Edwards lab and all communal equipment is held. Important points to note:
- Hoods need to be cleaned before and after each use with trigene
- Anything brought into the hood must be disinfected with trigene before it is brought in.
- Lab coats must be worn at all times in the tissue culture lab. This lab coat is kept in the lab.
- Hoods should be booked before hand, and as a matter of courtesy if you finish early, or think you will finish late you should let the next person know.
02/10/2009
I began to grow my cells with Alba. I began by creating my medium by adding L glutamine and foetal calf serum (FCS) to the medium provided. Then added this to cells from passage 23 and 32 which had been stored in liquid nitrogen. These cells were then transferred into small flasks and incubated at 37 degrees celcius.
I also attended the Edwards research group lab meeting and met the other members of the research group.
05/10/2009
Cells were passaged from P23 to P24 and P32 to P33. This was done by first aspirating the medium from the flask, washing with PBS buffer, aspirating again, then washing with TE to remove the cells from the matrix. The cells were transferred to larger flasks along with 10ml of the medium created previously. The cells were incubated again.
07/10/2009
The cells have been growing well but were not yet confluent so today I added fresh medium to feed the cells. I have incubated the cells again and will check on them again tomorrow and decide whether they need to be fed again or to be passaged again.
Today I had the building induction in the Biomedical Research Centre (BMRC) with Alba Warn. Alba took me through all health and safety procedures and took me around the lab showing me where each research group works, the communal areas, waste disposal, first aid kits and fire exits. Main points to note were:
Chief Technicians - Alba Warn & Jasmine Waters
Emergency Evacuation Procedures - Follow signs to Fire Exits
Alert others by striking 'break glass' boxes to set alarm
Assembly points: Across DMU loading bay for East Wing, and grass between BMRC and Sainsbury Centre for West Wing.
First aid information is on posters at each end of the lab
Labcoats to be worn at all times, along with gloves, goggles, face visor as appropriate (e.g. wear face visor and protective gloves when handling liquid nitrogen).
01/10/2009
Today was my induction to the tissue culture lab, again with Alba Warn. I was shown where all equipment for the Edwards lab and all communal equipment is held. Important points to note:
- Hoods need to be cleaned before and after each use with trigene
- Anything brought into the hood must be disinfected with trigene before it is brought in.
- Lab coats must be worn at all times in the tissue culture lab. This lab coat is kept in the lab.
- Hoods should be booked before hand, and as a matter of courtesy if you finish early, or think you will finish late you should let the next person know.
02/10/2009
I began to grow my cells with Alba. I began by creating my medium by adding L glutamine and foetal calf serum (FCS) to the medium provided. Then added this to cells from passage 23 and 32 which had been stored in liquid nitrogen. These cells were then transferred into small flasks and incubated at 37 degrees celcius.
I also attended the Edwards research group lab meeting and met the other members of the research group.
05/10/2009
Cells were passaged from P23 to P24 and P32 to P33. This was done by first aspirating the medium from the flask, washing with PBS buffer, aspirating again, then washing with TE to remove the cells from the matrix. The cells were transferred to larger flasks along with 10ml of the medium created previously. The cells were incubated again.
07/10/2009
The cells have been growing well but were not yet confluent so today I added fresh medium to feed the cells. I have incubated the cells again and will check on them again tomorrow and decide whether they need to be fed again or to be passaged again.
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