Hi all
earlier I have seen in some papers people go for DNA extraction and normal PCR using 16S rRNA primers for the identification of bacteria. However recently I have seen few papers particularly dealing with Uncultured “Candidatus” bacteria, researchers go for RNA extraction, reverse transcription RT-PCR and real-time RT-PCR ? Molecular biology experts can you please tell me …..
1. what’s the key advantage between the two ? is there any particular advantage of RT PCR for the identification of Uncultured “Candidatus” bacteria ?
2. Is it because of the possibility of “relative quantification” of the bacterium by real-time RT-PCR by targeting the 16S rRNA gene of the bacterium?
3. Is there any advantage when (RT PCR) used for uncultivable bacteria?
4. what is this Cycle threshold ? what is the significance of this in the above reaction ?
5. Also “The eukaryotic elongation factor 1 alpha from the host was used as a control of the RNA amount, and a good extraction was expected to give a Ct-value around 15 (the cycle threshold was set to 0.1). ? all results with Ct-values above 45 were considered negative !, what does it all mean?
My aim is just to identify the unculturable bacteria from tissues! Can I go for just normal PCR (16s rDNA) and sequencing the PCR products? Please
thank you
regards,
Hi, if the paper you mentioned is this one Article Candidatus Liberibacter americanus induces significant repro...
it's not identification of the candidatus, but it's RT-PCR used to analyze the gene expression difference between infected and non-infected citrus leaves.
2. no, it's not possible to differentiate bacteria using RT-PCR, and the term of "relative" is commonly used in gene expression analysis
4. cycle threshold is a term used to mention the "start" cycle where the amplification start exponentially
5. eukaryotic elongation factor 1 alpha is a gene that the authors used as house keeping gene (which is another term for normally expressed gene in the tissue thar are analyzed, and the expression data is used as a ratio to measure the expression of the gene of interest), the ct set to 15 is just a standard for them to measure the RNA isolation went good, which mean the isolated RNA concentration isnt too much or too low, which they mentioned that ct above 45 is considered negative (because it takes too long to amplify which also mean there is too few copy of RNA).
in my opinion, yes just do 16S PCR and sequence the product
hi Jonathan, thank you for your all response.
i am not referring to the paper exactly you mentioned, But of course I wanted to identify a Candidatus/uncultured bacteria. will go ahead with the 16S PCR and sequence the product....thank you again ...regards
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