I'm looking for a sequence or organism that I could use as a standard for 16S rRNA quantification by qPCR. And how I could validate the LD and LC.
I'm using TaqMan probes.
Thank you in advance.
What organism are you studying? That will make a big difference. If you are doing a eukaryote, make sure you pick a eukaryotic organism.
For bacteria, watch your GC content, and think about picking a species that is somewhat close. For bacterial communities, you can order mock communities with known concentrations/proportions.
Actually, I'm investigating about antibiotic resistance genes (ARGs) in different kind of environment samples (stool, sewage, soil). This genes are part of bacteria, bacteriophages and could be as free DNA. The objective of 16S rRNA quantification is normalize the data according with the total abundance of 16S genomic copies. But. To construct my qPCR standards, I use bacteria with cloned plasmids. In the case of 16S rRNA, I want to know about experiences quantifying this target to estimate total bacterial load in my samples.
this link useful
Article Ritalahti KM, Amos BK, Sung Y, Wu O, Koenigsberg SS, Löffler...
regards
Dear Jane Lucas , I am currently using two options:
1) I amplify a 16S rDNA fragment using primers 27F and 1492R using E. coli as the sample. Then, I purify the PCR product (approx. 200 ng/uL). I verify the presence of the amplimer by agarose gel electrophoresis and then I make the 10-fold dilutions. My qPCR amplimer is small (150 pb) and the performance was adequate according to the equipment manufacturers' specifications.
2) I designed a little fragment (approx 250 pb) of DNA that includes my amplimer and sent it to synthesize into a vector (plasmid pTOP blunt). The price is cheap (around USD 150) in Macrogen.
Both options work very well. Kind regards!
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