I want to measure abundance of Ammonia oxidizing bacteria using qPCR. I don't know how to construct my standard. Could you help me?
Hi, I am in busy field work, can you please follow the papers below:
Hai B, Diallo NH, Sall S, Haesler F, Schauss K, Bonzi M, Assigbetse K,
Chotte JL, Munch JC, Schloter M (2009) Quantification of key
genes steering the microbial nitrogen cycle in the rhizosphere of
sorghum cultivars in tropical agroecosystems. Appl Environ
Microbiol 75:4993–5000
Lindsay EA, ColloffMJ, Gibb NL,Wakelin SA (2010) The abundance of
microbial functional genes in grassy woodlands is influenced more
by soil nutrient enrichment than by recent weed invasion or
Töwe S, Albert A, Kleineidam K, Brankatschk R, Dumig A, Welzl G,
Munch JC, Zeyer J, Schloter M (2010) Abundance of microbes
involved in nitrogen transformation in the rhizosphere of
Leucanthemopsis alpina (L.) Heywood grown in soils fromdiffer
Good luck!
thanks a lot for your answer. I will read these papers
kind regards
Malchair,
Buy a few ATCC standard strains which oxidize ammonia and possess your gene sequence of interest; verify this before your purchase using the NCBI GenBank database. You’ll also need as many non-ammonia oxidizing bacteria as you can get your hands-on to validate specificity in your reaction. Standard strains are your friends in this line of work and are almost mandatory if you want your paper to survive the peer-review process.
Design several qPCR primer sets which are relevant to your gene sequence of interest. Screen each primer pair for amplification in each of your ATCC strains. Keep in mind that you will need to use ATCC strains (or a comparable standard). Otherwise, a natural isolate will need to be sequenced in-full for comparison to other strains; especially in terms of copy number.
Identify which primer pair is most efficient in qPCR amplification. If you want to multiplex the process, make sure that your amplicons are similar in length, GC composition, and that your primer Tm values are close to each other. Finally, you will need to optimize each stage of the polymerase chain reaction; duration, temperature, cycle quantity, etc.
I know this is going to sting a little but you need to construct a growth curve for each strain. Know exactly how many cells you inoculate into a broth culture, how fast the tube is agitated in rotation, how many hours it will take to reach stationary growth, and the relationship between spectrophotometric optical density absorbance and CFU growth on solid media. If you want a quantitative standard curve, this is paramount.
From here, fix each broth culture to a specific optical density absorbance at a specific (early) stationary growth phase. Validate several times that you are precisely sure how many cells you have in your tube. From here its all very basic. Dilute tenfold, extract DNA via lysis boiling or kit, run the qPCR and build your curve based on initial cell quantities and cycle threshold values.
You will likely see variation in cycle threshold trends between strains due to differences in copy number. If you don't you're ready to publish. Don't forget to include melting curve data. You need to make sure that you verify that primer dimerization is a non-issue. If the standard curves are significantly different between strains, you'll need to consider multiplexing with species-specific primers; or at least a separate reaction with such primers.
Have at it.
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