Hi Everyone,
I am trying to check for Gene Expression in one of the Bacteria(prokaryote), so i had planned for RNA-Sequencing i.e. Transcriptome Analysis.So,
1.Is it possible to isolate m-RNA with or without commercial kits, how about the efficiency of the isolated product,any procedure available for isolation of m-RNA from Prokaryotes!
2.How to proceed with the isolated product to construct cDNA, should i follow the conventional RT- PCR, which are the Primers/Enzyme can be used ?
3.Once cDNA has been constructed, can i sequence it for further analysis .
no mRNA in procaryotes however you can use random primers more specifically hexamers
You could start by reading literature about similar experiments. The materials&methods section should give you a first idea about possible approaches. Unfortunately, I am not sure about your intention. Are you interested in comparing gene expression between different samples or is the RNA-seq experiment planned for annotation purposes? The appropriate method depends on your research question. Could you please provide some more details about your project?
Sorry: what is this no "mRNA in procaryotes" you perhaps mean no poly-adenylation? The first demonstration of the existence of mRNA was in E. coli (the first definitive experiment, there were some adumbrations after phage infection and enzyme induction, always in E. coli).
Brenner, Jacob & Meselson, "An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis," Nature 190 (1961) 576-80.
Thanks for the reply, Sandeep Raj Sir, Prokaryotes has an m-RNA , i didnt get that what you are about to convey.,
#Boas pucker, what am interested is to isolate m-RNA from prokaryotes, construct cDNA and sequence it , hope you get that !
Well I misunderstood your sentence "no mRNA in procaryotes however you can use random primers more specifically hexamers" to mean "there is no mRNA". Whatever you meant the it is now clear you did not mean "there is no"
Claudio
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