Sep 18, 2024
Quantification of 18S rRNA Gene Copies Using ddPCR (probe-based assay: FungiQuant F-FungiQuant Prb-FungiQuant R)
- 1Soil and Water Research Infrastructure
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: eva.petrova@bc.cas.cz
Protocol Citation: Eva Petrova, Roey Angel 2024. Quantification of 18S rRNA Gene Copies Using ddPCR (probe-based assay: FungiQuant F-FungiQuant Prb-FungiQuant R). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo5wr9l4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 30, 2020
Last Modified: September 18, 2024
Protocol Integer ID: 36416
Keywords: Absolute quantification, ddPCR, 16S rRNA
Abstract
The protocol is dedicated to evaluation of 18S rRNA fungal gene copy number using Droplet Digital PCR technology (ddPCR) from Bio-Rad company. This is the up-to-date modification and improvement of clasical probe based qPCR assay.
For the assay, we are using universal fungal 18S rRNA primers and a probe that showed broad-coverage and favourable quantitative parameters (adapted from Liu et al., 2012):
FungiQuantF 5'- GGR AAA CTC ACC AGG TCC AG -3' , targets S. cerevisiae 1199-1218,
FungiQuantPrb* 5'- TGG TGC ATG GCC GTT -3', targets S. cerevisiae 1269-1283,
FungiQuantR 5'- GSW CTA TCC CCA KCA CGA -3', targets S. cerevisiae 1532-1549.
* Probe must be dual-labelled either with 5’-6-FAM, 3’-BHQ1 or any other valid combination.
The expected amplicon length is 351bp.
Note
Even thoughthe assay is very robust and can theoretically match 1,057 fungi genera and 2,465 fungal species evaluated by in silico testing, one has to keep in mind that some exceptions persist. For example, the Mucoromycotina, Chytridiomycota, and Kickxellomycotina genera have only partial sequence matches, and Microsporidia, together with Entomophthoromycotina subphyla, do not possess any perfect matches to FungiQuant.
Attachments
Guidelines
- The crucial steps that can influence the final assay results a lot are precise pipetting, mixing and dilutions!
- Keep in mind that ddPCR technigue do not posses as wide dynamic concentration range as qPCR does. You can easily "overload" the reaction with too much template DNA putting in. In that case you will see only positive droplets at the end and no negative and system will not be able to calculate copy number for them. As a consequence, you usually have to dilute your template DNA more than for qPCR experiments. Ideally fit inside the range of 101 - 104 coppies of target gene. That is why we usually test several dilutions of few samples in advance to see "where we are".
- One have to also keep in mind that using ddPCR technology you must work within the format of eight. If you do not fulfil all the wells in a cartridge you still have to put the reagencies inside the empty wells as well. So, think economically before you start.
Materials
MATERIALS
ddPCR Supermix for probes (no dUTP)BioRad SciencesCatalog #1863024
PCR Plate Heat Seal foil piercableBio-rad LaboratoriesCatalog #1814040
ddPCR 96-well platesBio-rad LaboratoriesCatalog #12001925
Automated Droplet Generation oil for ProbesBio-rad LaboratoriesCatalog #186-4110
Protocol materials
ddPCR Supermix for probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863024
Materials
PCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
In Materials and 2 steps
ddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925
Materials, Step 2
Automated Droplet Generation oil for ProbesBio-Rad LaboratoriesCatalog #186-4110
Materials, Step 7
ddPCR™ Supermix for Probes (No dUTP)Bio-Rad LaboratoriesCatalog #1863023
Step 2
Safety warnings
Protect probe from light.
Before start
Take all the reagencies out of a freezer and let them temperate to room temperature.
ddPCR reaction mixture
ddPCR reaction mixture
20m
20m
All reagencies must be equilibrated to RT (do not keep them on ice). Mix each of them properly before use.
20m
| Reagent | Final conc. | 1 tube (22 μl) | plate (22 μl x 100) | |
| PCR H2O | 6.6 | 660 | ||
| ddPCR Supermix for for Probes (no dUTP) | 1x | 11 | 1100 | |
| FungiQuant F (20 μM) | 0.9 μM | 1 | 100 | |
| FungiQuant R (20 μM) | 0.9 μM | 1 | 100 | |
| FungiQuant Prb (10 μM)† | 0.2 μM | 0.4 | 40 | |
| Template | 2 | 2 x 100 |
† Probe must be dual-labelled either with 5’-6-FAM, 3’-BHQ1 or any other valid combination.
Prepare the master mix according to the number of samples and mix several seconds by vortexing. Transfer mix into 96-well plate à 20 µl.
20 µL master mix per well
ddPCR 96-well platesBio-rad LaboratoriesCatalog #12001925
ddPCR™ Supermix for Probes (No dUTP)Bio-rad LaboratoriesCatalog #1863023
Note
Tip: use a mechanical or electronic dispenser (for ex. Multipette, Pipettman, or multichanel pipette) during this step to speed up the work.
Add 2 µl of your DNA sample into each well
2 µL of examined DNA per well
Seal the plate (180°C, 5 s) with pierceable aluminium foil.
PCR Plate Heat Seal foil piercableBio-rad LaboratoriesCatalog #1814040
00:00:05 sealing at 180°C
Let the foil cool down and mix the plate vigorously by vortexing 30 s - 1 min.
00:00:30 vortexing
Droplets generation by AutoDG
Droplets generation by AutoDG
Put all the staff (cartridges, tips, sealed plate with samples and empty 96-well plate in cooling stand) in corresponding amount inside the AutoDG machine (Bio-Rad).
Note
Per one sample there is a need of two pipette tips!
This is an example how it should look like inside the AutoDG before strating droplets generation.
Make sure there is a right oil bottle (Automated Droplet Generation oil for Probes) connected to the system.
Automated Droplet Generation oil for ProbesBio-rad LaboratoriesCatalog #186-4110
Oil type (in use) is also displayed on AutoDG touch screen
Choose a position of samples on touch screen and start dropplets generation. Wait after its finished.
Note
System will you announce automatically about success or failure of droplets creation after procedure is finished. Nevertheless, every time make also a visual inspection of droplets. Two separated phases should be visible. Upper part with dropplets and lower clear oil phase.
This is how it should look like inside the AutoDG after droplets generation.
a plate with two visible separated phases - upper phase contains droplets
Take the plate with droplets out of the machine and seal it with pierceable aluminium foil (170°C, 3s).
PCR Plate Heat Seal foil piercableBio-rad LaboratoriesCatalog #1814040
00:00:03 sealing at 170°C
Put immediately the sealed plate into PCR cycler.
Note
Droplets are unstable at this stage. Proceed to next step as fast as possible. After PCR droplets become stable and can be kept at fridge for some time (one day) before measurement.
Clean AutoDG machine, waste used consumables.
PCR program
PCR program
3h
3h
1. 95◦C –10′
2. x 40 {
a. 94◦C – 30′′
b. 60◦C – 2′
}
3. 98◦C –10′
4. 10◦C –hold
Set ramp rate for each step to 2°C/sec!
Set reaction volume to 40ul.
40 µL reaction volume
Note
- After run is finished check if there are still two phases present
- Let the plate cool down (droplets will not be so sticky and will be more easy to analyse).
Droplets reading
Droplets reading
Note
Switch on the reader 30 min before measurement.
Droplet reader with a plate after PCR already placed inside the metal holder
Set up the QuntaSoft experiment as follows:
Exp. type: Absolute quantification (ABS)
Supermix: ddPCR Supermix for Probe
Target1: Ch1 (for FAM labeled probes)
Define the position of ech sample.
Check the levels of reader Oil and waste - green control (bottles are physically accessible from machines left side).
Note
If the instrument was not in use longer than week it has to be Primed first (oil flushed).
Start measurement.
Note
After it finishes go over the results and see how many droplets were executed for each sample. To get reliable results total droplet count should be above 12.000. We usually have got 18.000 - 20.000 droplets analyzed per sample.
An example of total droplet counts, including positives and negatives together, for several environmental samples.
Analysis
Analysis
Set up the treshold just above negative control sample in order to distinguish positive (containing PCR products) from negative (do not contain any PCR product) droplets.
Note
Quanta software will automaticaly calculate copy number of target gene for each sample by Poisson distribution algorithm. For that calculations at least some of the negative droplets are necessary. If the sample contains only positive population it can not be evaluated.
An example of 18S Fungi copy numbers data analysis. Last two samples on the left graph are negative (NTC) samples by which a treshold was set up.
Export .csv file with concentrations (copies/ul) that are posessing you number of copies in 1ul of reaction. To obtain number of copies in 1ul of your input DNA you have to recalculate:
no. of copies in 1ul of input DNA = (concentration value x 22)/ volume of DNA