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16SrRNA_Quantification.md

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qPCR-based detection and quantification of bacteria

Materials and Equipment

  • iTaq™ Universal Probes Supermix (BioRad 1725132)
  • CFX384 thermocycler (Biorad)
  • 384 Plates for qPCR (Biorad #HSP3865)
  • Optically clear Plate Seals (Biorad Microseal ‘B’ #MSB1001)
  • 10x Assay mix (prepare 2µM of each oligo below in nuclease-free water)
  • 10-fold dilution series of gDNA (from any bacteria). Use the QuBit DNA concentration to calculate 16S copy number based on genome size to genome copy number and rRNA copy number. For example: 1ng of E. lenta DSM 2243 DNA ~ 2.7E5 genome copies and the 16S rRNA copy number is 3, so the first point on a 10-fold dilution series prepared from 10ng/µL gDNA is 8.1E5 16S rRNA copies/µL. Alternatively a full length 16S rRNA amplicon an be used instead for the standard but be sure to dilute to at least 1e8 copies/ul before loading on standard curve.
  • Extracted gDNA and knowledge of the original weight/volume extracted and the final elution volume. Check the gDNA using a nanodrop and if there is any indiciation of suspected ethanol carryover, dilute all samples 10x before running.

Oligos

Oligo Sequence Stock [µM] Working [µM] Final [nM]
891F TGGAGCATGTGGTTTAATTCGA 100 2 200
1003R TGCGGGACTTAACCCAACA 100 2 200
1002P [6FAM]CACGAGCTGACGACARCCATGCA[BHQ1] 100 2 200

Setting up qPCR RXNs

The master mix we are using has an antibody-mediated hot-start, and as such can be set up at RT in the PCR flow-hood in a 384 well plate. If quantification is desired, the reactions should be set up in triplicate.

Reaction mixture

Component [stock] µL/rxn [final]
iTaq Supermix 2X 5 1X
10X assay mix 2µM 1 200nM
Water - 2 -
DNA template variable 2 varable

After setting up the plate, breifly centrifuge to mix and remove air bubbles before transfering to the CFX384.

Cycling Parameters

Cycle Temperature (˚C) Time
Initial Denaturation 95 5min
40 cycles:
Denature 95˚C 5 sec
Anneal/Extend 60˚C 15 sec
Hold 4˚C Hold 0 sec

When setting up the plate using the BioRad software, select the 384 plate template with all channels (not the FAM/SYBR only). Using the plate editor, change your standard curves from unknown, to standards and enter the copy number you have calculated.

Interpretation

To convert from the assay concentration to the 16S copy per gram or mL it is essential to know the original mass/volume that was extracted. Assuming the DNA was extracted from 100 mg of feces and the final elution volume during the DNA extraction was 50 ul, and a 10x dilution was performed before the assay see below:

5e8 16S copies/ul (from qPCR assay) * (50 ul elution volume / 4 ul template in rxn) * (10 Xdilution) / (0.1g) = 6.2e11 16S copies/g.