Nov 23, 2020

Public workspaceQuantification of 16S rRNA Gene Copies Using ddPCR (EvaGreen-based assay: 338F-805R) V.3

DOI
https://dx.doi.org/10.17504/protocols.io.bpy6mpze
Quantification of 16S rRNA Gene Copies Using ddPCR (EvaGreen-based assay: 338F-805R)
  • Roey Angel1,
  • Eva Petrova1
  • 1Soil and Water Research Infrastructure
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: [email protected]
Roey Angel
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DOI: https://dx.doi.org/10.17504/protocols.io.bpy6mpze
Protocol Citation: Roey Angel, Eva Petrova 2020. Quantification of 16S rRNA Gene Copies Using ddPCR (EvaGreen-based assay: 338F-805R). protocols.io https://dx.doi.org/10.17504/protocols.io.bpy6mpzeVersion created by Roey Angel
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: November 23, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44798
Keywords: digital droplet PCR, 16S rRNA gene, bacteria, quantification of 16s rrna gene copy, rrna bacterial gene, classical bacterial enumeration qpcr, rrna gene copy, bacterial gene, using ddpcr, ddpcr, pcr, rrna, droplet digital pcr technology, using droplet digital pcr technology, transcript copy number, based assay, bio,
Abstract
This protocol describes how to quantify 16S rRNA bacterial gene or transcript copy numbers using Droplet Digital PCR technology (ddPCR) from Bio-Rad. This is an up-to-date modification of a classical bacterial enumeration qPCR-assay. This assay uses the EvaGreen™ chemistry. The primers are taken from Yu et al. (2005).


Note
Advantages of ddPCR over qPCR Among the biggest advantages of the ddPCR technique are its high sensitivity (down to one molecule of target gene presented in input DNA) and low sensitivity to enzymatic inhibitors. Moreover, because it is an absolute and direct quantification technique, no external standard is needed for evaluation.


Citation
Yu Y, Lee C, Kim J, Hwang S (2005). Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnology and bioengineering.

Attachments
Icon for MSDS-EvaGreen.pdf
MSDS-EvaGreen.pdf
32KB
Guidelines
  1. Just like for qPCR, careful and precise pipetting, and adequate mixing and dilutions are crucial to the success of the assay.
  2. Keep in mind that ddPCR works with a lower dynamic concentration range compared to qPCR does.
  3. One can easily overload the reaction with too much template DNA because ddPCR requires that a certain proportion of the droplets remain empty. Ideally, a ddPCR reaction should contain between 101 - 104 copies of the target gene. If the expected copy-number range cannot be assumed in advance it is advisable to prepare several dilutions of the sample in parallel and analyse them together.
  4. The ddPCR Droplet Reader processes the samples in batches of 8. Therefore, even if the total number of samples is not a multiplication of 8 all the columns in the plate must be filled with reagents.
Materials
STEP MATERIALS
ReagentddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925
ReagentQX200™ ddPCR™ EvaGreen SupermixBio-Rad LaboratoriesCatalog #1864033
ReagentPCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
ReagentAutomated Droplet Generation Oil for EvaGreenBio-Rad LaboratoriesCatalog #1864112
ReagentPCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040



Protocol materials
ReagentQX200™ ddPCR™ EvaGreen SupermixBio-Rad LaboratoriesCatalog #1864033
ReagentPCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
ReagentAutomated Droplet Generation Oil for EvaGreenBio-Rad LaboratoriesCatalog #1864112
ReagentPCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
ReagentddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925
Safety warnings
See the regulations of your institute for proper handling and disposal of DNA-intercalating dyes. The MSDS of the EvaGreen dye is enclosed in this protocol.
Before start
Take all the reagents out of the freezer and allow them to reach room temperature.
Primers
The assay uses the following universal 16S bacteria primers:
NameDirectionSequenceTarget region1
BAC338FFACT CCT ACG GGA GGC AG338-354
BAC805RRGAC TAC CAG GGT ATC TAA TC785-805

     1. Relative to E. coli SSU rRNA gene
PCR mixture
40m
All reagents must be equilibrated to TemperatureRoom temperature (do not keep them on ice). Mix each of them properly before use.

20m
Critical
ReagentFinal conc.1 tube (22 μl)plate (22 μl x 100)
PCR H2O 8.6860
QX200 ddPCR EvaGreen Supermix1x111100
BAC 338F (10 μM)0.1 μM0.220
BAC 805R (10 μM)0.1 μM0.220
Template 22 x 100


Prepare the master mix according to the number of samples (incl. at least one NTC sample) and mix for several seconds by vortexing or pipetting. Transfer mix into 96-well plate à Amount20 µL .
ReagentddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925

ReagentQX200™ ddPCR™ EvaGreen SupermixBio-Rad LaboratoriesCatalog #1864033

Note
Tip: use a mechanical or electronic dispenser (e.g. Multipette, Pipettman, or a multichannel pipette) during this step to speed up the work.

10m
Pipetting
Add Amount2 µL DNA template into each well.

5m
Seal the plate (Duration00:00:05 Temperature180 °C ) with a pierceable aluminium foil.
ReagentPCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040

Equipment
PX1 PCR Plate Sealer
NAME
Plate Sealer
TYPE
Bio-Rad
BRAND
1814000
SKU
https://www.bio-rad.com/en-at/sku/1814000-px1-pcr-plate-sealer?ID=1814000
LINK


PX1 Plate Sealer set-up

5s
Let the foil cool down and mix the plate vigorously by vortexing for Duration00:00:30 -Duration00:01:00
Mix
Droplet generation using AutoDG
1h
Place the cartridges, tips, sealed plate with samples and an empty 96-well plate into their appropriate positions in the QX200 AutoDG Droplet Digital PCR System (Bio-Rad).

Note
Note: Two pipette tips are needed for each sample!
This is how it should look like inside the AutoDG before starting droplets generation

Equipment
Automated Droplet Generator
NAME
Droplet Digital PCR System
TYPE
Bio-Rad
BRAND
1864101
SKU
https://www.bio-rad.com/en-at/sku/1864101-automated-droplet-generator?ID=1864101
LINK

40m
Make sure that the correct oil bottle—Automated Droplet Generation oil for EvaGreen—is connected to the system.


ReagentAutomated Droplet Generation Oil for EvaGreenBio-Rad LaboratoriesCatalog #1864112

Note
The droplet generation oil is prone to expire about one year after opening. Replace the oil if it has turned milky.

Mark the position of the samples in the plate on the touch screen and press "START" initiate the droplet generation.

A confirmation window will appear on the screen before the procedure starts. Make sure you have chosen the right positions of the samples.
Note
After the droplet generation is finished, the system will you automatically display a message about the success or failure of the procedure. Nevertheless, even after a successful run, it is advisable to inspect the wells and ensure that two separated phases are clearly visible. Upper part with droplets and lower clear oil phase.
This is how it should look like inside the AutoDG after droplets generation
The 96-well plate after droplet generation: two phases are visible in each well containing a sample

Take the plate with droplets out of the AutoDG and seal it with pierceable aluminium foil (Temperature170 °C , Duration00:00:03 ).
ReagentPCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
Immediately place the sealed plate into PCR cycler (see below) and initiate the reaction.

Note
The droplets are unstable at this stage. Proceed to the next step as soon as possible. Following PCR, the droplets become stable and can be kept at Temperature4 °C for some time (up to 24 hours) before measurement.

Critical
Clean the AutoDG and discard used consumables.
PCR program
1.  Temperature95 °C Duration00:05:00
2.  x 5 {
       a.  Temperature95 °C Duration00:00:30
       b.  Temperature60 °C Duration00:02:30 ′ (Temperature-1 °C each step)
      }
3. x 35 {
       a.  Temperature95 °C Duration00:00:30
       b. Temperature55 °C Duration00:02:30
      }
4. Temperature4 °C Duration00:05:00
5. Temperature90 °C Duration00:05:00
6. Temperature10 °C hold

Settings:
  1. Set the ramp rate for each step to 2°C/sec.
  2. Set the reaction volume to Amount40 µL .
Note
  • After the run is finished check if there are still two phases present.
  • Let the plate cool down before downstream measurement
  • To maximise the droplet count, leave the plate overnight in a fridge before processing the samples in the QX200 Droplet Reader. This is because the droplets tend to stick together after the PCR step, but loose this tendency after prolonged co

Equipment
T100™ Thermal Cycler
NAME
Thermal Cycler
TYPE
Bio-Rad
BRAND
1861096
SKU
https://www.bio-rad.com/en-at/product/t100-thermal-cycler?ID=LZJU45E8Z
LINK

3h
PCR
Droplet reading
30m
Put the plate into a metal holder, place them together into QX200 Droplet Reader.

Note
Switch on the reader 30 min before measurement.

Droplet reader with a plate after PCR already placed inside the metal holder

Equipment
X200™ Droplet Reader
NAME
Droplet Reader
TYPE
Bio-Rad
BRAND
1864003
SKU
https://www.bio-rad.com/en-at/sku/1864003-qx200-droplet-reader?ID=1864003
LINK

1h
Imaging
Set up the QuntaSoft experiment as follows:


Exp. typeAbsolute quantification (ABS)
SupermixEvaGreen ddPCR Supermix
Target1Ch1

Define the position of each sample.
Check the levels of reader Oil and waste - green control (bottles are physically accessible from the left side of the device).


Note
If the instrument was not in use for longer than one week, it has to be primed first (oil flushed).


Start the measurement.

Note
After the count is finished go over the results and check how many droplets were counted for each sample. To get a reliable count the number of droplet should be above 12.000. On average, droplet counts range from 16.000 to 18.000.
An example of total events = droplet counts (positives and negatives together) for ten samples.


Analysis
Set the threshold just above the negative control sample in order to distinguish positive (droplets containing PCR products) from negative (empty) droplets.

Note
QuantaSoft will automatically calculate a copy number of the target gene for each sample using its Poisson distribution algorithm. For these calculations, a certain portion for the droplets must be negative. If the sample contains only positive droplets it cannot be evaluated properly (see an example below).
An example of 16S Bac copy numbers data analysis. The right-most sample is a negative-control sample (NTC), according to which a threshold was set up. Sample 4 (well D10) shows an overloaded sample with an insufficient number of negative (empty) droplets. The quantification for this sample is inaccurate and the sample should be repeated with a higher dilution.

Software
QuantaSoft Analysis Pro
NAME
Windows 7 or Windows 10, 64 bit 1.7.4
OS
Bio-Rad Laboratories
DEVELOPER
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10047467.pdf
SOURCE LINK

Analyze
Export a CSV file with concentrations (copies µl-1). To obtain the number of copies in 1 µl of template DNA use the following formula:
no. of copies in 1 µl of template DNA = ddPCR conc. x 22 / volume of template DNA

Citations
Yu Y, Lee C, Kim J, Hwang S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction.