I tried to amplify the glutymal-tRNA sequence (more than 1500bp amplified to guarantee its sequence included) from the genome of corybacterium glutamicum and cloned in an expression vector. How can I know whether it is correctly synthesized ? Any better way to detemine the level of glutymal-tRNA in bacteria?
HI
You have the choice between hybridization (Norhtern blot, or Chip) or amplification (RT-PCR, RNA-Seq). Since tRNAs are fully of base modifications that block reverse transcription, I definitely recommend hybridization-based methods. Here are two relevant papers (google or pubmed): PMID: 15640439, PMID: 16387656
Hey,
I agree with the suggestions by Mark. qRT-PCR is unfortunately not the best choice, due to the modifications. However, one can try and sometimes it still works. The problem is to design primers which bind uniquely to your tRNA of interest. You have to make sure that the primers bind within the 70-80 nts (whatever the length of your mature tRNA is) of your tRNA. And you have to do the reverse trasncription at relatively high temperatures, because of the intense secondary structure.
A better and still relatively easy to perform method is the Northern blot, as suggested by Mark (depends on the concentration of the tRNA. Is the concentration very low one has to use radioactive probes). Another way is to perform microarrays, established and published by Tao Pan (University of Chicago; Dittmar KA, Goodenbour JM, Pan T, Tissue-specific differences in human transfer RNA expression, PLoS Genet. 2006 Dec;2(12):e221.).
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