Hi,
Iam starting a new project in which Iam silencing down two genes by lentivirus. Lets say gene x and Y. I have two single and one double knock downs. I want to study the expression of the genes involved in cancer progression, for this I am planning to do RNA sequencing for the single and double knockdown.
My question is, if I do RNA seq, will I be able to compare the gene expression levels of say MAPK pathway in the control and the knockdowns. If Iam not right, please suggest me a method, were I can be able to study the genes involved in cancer progression (control Vs knockdowns).
Thanks
jaya
I guess that you have already checked that X and Y are knocked down (RNA and protein) and that you have a lentivirus control with a negative control. As a start, I would suggest to check the effect on putative target RNA or protein, e. g. by qPCR or Western. You may later do whole transcriptome analyses but since tumor supression will affect many downstream genes it will be helpfull to start with a few targets and controls. This may also help to define a good time point for RNA extraction.
Also in order to study the genes involved in cancer progression I suggest you to take all your significant genes and use for example DAVID (https://david.ncifcrf.gov/) in order to check with pathways were affected. Then you can take those pathways implied in your disease and check how differential expression affects them
Hi Jaya,
If you're solely interested in the involvement of a certain pathway (e.i. MAPK), then a targeted approach using specific qPCR primers for components of that pathway is the way to go. However, if you want to take an unbiased approach and investigate differential expression between the different isogenic cell lines then RNAseq would yield much richer data. When performing RNAseq consider replicates to obtain statistical significance. There are freely available tools for non-bioinformatician with corresponding tutorials you could use. Seqmonq and Altanalyze are tow examples. Finaly, it's always advisable to discuss with a bioinformatician for help with design of the experimental procedure.
Najim
http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/
http://www.altanalyze.org/
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology