Bioinformatics

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.