Ludlow
SOP Shay/Wright lab
1/27/13
Materials
Droplet Digital TRAP
Reagents and Equipment –
1. Cell pellets – 25,000 to 100,000 cells
2. RNase/DNase free dH2O (Ambion)
3. Ultrapure BSA (Ambion)
4. 50xdNTP mix (2.5mM of each dATP, dCTP, dGTP, dTTP)
5. 10M TS primer (HPLC purified)
6. 10M ACX primer (HPLC purified)
7. NP-40 lysis buffer (RNase/DNase-free): 10 mM Tris-HCl, pH 8.0; 1 mM MgCl2; 1
mM EDTA; 1% (vol/vol) NP-40; 0.25 mM sodium deoxycholate; 10% (vol/vol)
glycerol; 150 mM NaCl; 5 mM -mercaptoethanol; 0.1 mM AEBSF (4-(2Aminoethyl)benzenesulfonyl fluoride hydrochloride)
8. 10 TRAP buffer (RNase/DNase-free): 200 mM Tris-HCl, pH 8.3; 15 mM MgCl2;
630 mM KCl; 0.5% Tween 20; 10 mM EGTA
9. twin-tec 96 well plate – eppendorf 951020362 (Fisher)
10. Easily pierced foil – less than 42m (Thermo Scientific AB0757).
11. LTS XLT Rainin pipet tips
12. Droplet generator cartridges (DG8), Biorad 186-3008
13. Droplet generator oil, biorad 186-3005
14. Droplet cartridge gaskets (DG8), biorad 186-3009
15. QX200 evagreen ddPCR supermix v2.0 (10028083 – for reference only not for
sale).
16. 8 channel pipet, Rainin LTS lite (1-20L, and 20-200L).
17. Single channel pipettes, Rainin LTS lite
18. Reagent troughs.
19. Thermocycler capable of fitting the 96 well skirted plates and adjusting the
temperature ramp rate (i.e., Biorad T100).
20. Droplet reader oil – second generation (v2.0).
21. Droplet reader (QX200) capable of reading Evagreen chemistry
Reagent setup –
All typical PCR precautions should be taken when setting up ddPCR TRAP reactions.
Further one should try to maintain an RNase/ DNase free environment since
telomerase relies on an RNA template molecule, RNases will significantly impact
telomerase enzyme activity detection.
Procedure –
Sample preparation (preparation of lysates)–
Approximately 45min to 65min.
1. Harvest cells
Harvest 25,000–100,000 cells into a DNase-, RNase-free 1.5-ml microfuge tube.
Pellet cells by centrifugation in a tabletop centrifuge at 3,000g for 5 min at room
temperature (18–25 1C). Carefully discard the supernatant. It is not necessary to
wash the pellet, but ensure that all residual liquid is removed. Proceed to Step 2 if
samples are ready for lysis.
NOTE – For ddTRAP 50,000 cell pellets have produced the most reliable and
reproducible results. This will be expanded upon below.
Pause point – Samples can be flash frozen and stored at -80ºC until ready for lysis.
2. Cell lysis
Suspend cells at a concentration of 1250 cell equivalents per microliter or less in
NP-40 lysis buffer and incubate on ice for 30min (e.g. 50,000 cells in 40L = 1250
cell eqs./ L).
3. Telomerase extension reaction
1. Keep lysates on ice. Lysates can be stored at -80ºC for up to 1 year, freeze thaws
should be avoided.
2. Prepare a telomerase positive cell line control with all assays (HeLa, H1299,
MCF7, A549 etc.). Making a dilution curve is not necessary for ddTRAP but can be
performed as an additional control.
3. Prepare a lysis buffer control to test for contamination in the lysis buffer.
4. RNase or heat-treated negative controls can also be included but are not
necessary for ddTRAP.
5. Prepare a telomerase extension master mix (n+2). The final volume of the
extension reaction is 50L. Prepare a master mix containing 1x TRAP buffer, 1x
dNTP mix (50x = 2.5mM thus 0.05mM = 1x), 0.2M TS primer per sample, 20g*mL1 BSA, and dH2O to 49L per sample. Add 1L of cell lysate to 49L of extension
master mix. If cells lysed at 1250cells/uL then 1250cells/50L = 25 cells/microliter
final concentration of extension. See example below:
Reagent
10x TRAP
buffer
2.5mM dNTP
50mg/mL BSA
10M TS
1250 cells / L
Lysate
dH2O
Volume (L)
for 1x
5
Volume for
10x
50
Volume for
20x
100
Volume for
30x
150
1
0.4
1
1
10
4
10
-
20
8
20
-
30
12
30
-
41.6
416
832
1248
Molecules of active telomerase/
cell
6. Incubate samples for 40min at 25ºC followed by a telomerase inactivation step of
95ºC for 5min, and then hold at 4ºC.
NOTE – The extension time was empirically determined for ddTRAP. We observed
increased detection of telomerase molecules per cell with increased extension time.
90
80
70
60
50
40
30
20
10
0
0
50
100
150
Duration of extension reaction (min)
Figure 1. Empirical determination of telomerase extension duration. TRAP lysates
from H1299 cells (in duplicate) were incubated for 7.5, 15, 30, 60 and 120 minutes
in telomerase extension reactions followed by ddTRAP.
7. Pause point - Extension products can be stored at -20ºC until analyzed in
ddTRAP.
4. Droplet digital PCR (or ddTRAP). –
1. Using good PCR practices set up the ddTRAP PCR reaction as follows: 1x
Evagreen ddPCR super mix v2.0 (Biorad), 50nM TS primer, 50nM ACX primer, 50cell
equivalents or less of extension product and dH2O to 20uL per sample.
Note: Ten percent (10%) extra volume of each reagent should be used so that the
final reaction volume is 22L to help prevent volume shortage when pipetting into
the droplet generator cartridge. You must make samples in intervals of 8 (see note
in step 4 below). This is a hotstart taq so all steps should be performed at RT.
Performing this set up on ice may increase solution viscosity which will affect
droplet formation.
For example:
Reagent
2x Evagreen
ddPCR supermix
(v2.0)
10M TS
10M ACX
25 cells/L
extension product
dH2O
Volume (L) 1x
11
Volume (L) 10x
110
Volume (L) 20x
220
0.11
0.11
2
1.1
1.1
-
2.2
2.2
8.8
88
176
2. Set up droplet generation (DG) cartridge by first loading 20L into the sample
well in the cartridge.
3. Load 70L of v2.0 droplet generation oil into the oil well.
NOTE: ORDER IS ESSENTIAL. The oil is a fluorinated making it heavy which if
loaded first causes the oil to flood the microfluidics and create a situation of poor
droplet formation. Thus it is essential that you load your sample first then the oil for
proper droplet formation.
4. Attach a DG cartridge gasket.
NOTE the droplet generation machine works only with a full cartridge of 8 full
wells. The machine stops when air (i.e., an empty well) is encountered. Thus
it is essential that you calculate your reactions to account for ‘extra samples’
as a control solution is not available yet for this machine. If you only have 12
samples you must still set up 16 reactions, just four of the reactions will be
blanks or no template controls (NTC). If you do not want to waste primer you
can generate blanks by mixing 11L ddPCR supermix with 11L of water per
open well.
5. Place assembled droplet cartridge into droplet generation machine.
6. Remove cartridge from the DG machine once droplet generation cycle is
completed (about 90sec).
7. Remove gasket gently.
8. Using the 8 channel pipet remove ~42L of emulsion (droplets) from the droplet
wells in the cartridge and place into a 96 well plate (Twin tec). This step is critical.
You must not burst the droplets or you will have poor results. Move slowly and
consistently. Do not repeat pipet or completely aspirate the droplets from the pipet
as this will enhance droplet breaking.
Repeat the above steps until all samples are loaded into the PCR plate.
9. Once all samples are loaded into the 96well PCR plate, the plate must be foil
sealed to prevent evaporation and light exposure of the emulsion (droplets). Place a
heat sealing foil on top of the plate (adhesive side down, all foils are oriented in the
packaging such that the adhesive side is face down, do not mix this up or you will
adhere the foil to the plate sealer. If this does happen, DO NOT PANIC! Turn off the
heat sealer and allow it to cool to room temperature or at least until you can touch it
safely. Use only water on the heat sealer, other chemicals will void the warranty
and potentially damage the sealer.). Seal the plate for 2sec at 165ºC, rotate the
plate 180º and repeat sealing for another 2sec. This ensures that all sides of the foil
are sealed.
NOTE: The un-used wells in the plate can be used in future runs. Thus, we save our
plates, just remove the foil carefully and add new samples and re-seal (this works
once or twice). Second, you can use thin foils (21m) that can be sealed over.
4. PCR step.
1. Load the 96 well plate into the thermocycler and close the lid. We use the T100
(Biorad).
NOTE: The C1000 96 well block fits the plates as well but we have not tested the
consistency of the assays between cyclers.
PCR reaction conditions – NOTE ALL ramp rate between temperature steps must be
set to 2.5ºC/sec in order to achieve even heating of the reaction mixture.
95ºC for 5min (activation of Taq poly).
40cycles of:
95ºC for 30sec
54ºC for 30sec
72ºC for 30sec
22ºC hold.
Timing = 1hr 45min for PCR reaction.
With set-up of full plate – 2.5 hrs maximum.
5. Measuring the fluorescence on the droplet reader
1. Once the thermocycler indicates that the run is complete and the samples have
reached 22ºC the plate can be removed from the machine.
Note – droplets should be read as close as possible to PCR completion. We have
observed fluorescent drift (i.e., loss of signal to noise ratio) if the plate is left o/n at
22ºC. Thus one must time their plate reading accordingly. Each 96 well plate takes
~3 hrs on the droplet reader.
2. Load the 96well plate into the plate holder matching well ‘A1’ with the ‘unnotched’ corner.
3. Close the reader lid.
4. Open ‘quantalife’ software on the desktop.
5. Double click well A1 in the plate template. This will change the upper part of the
screen to display ‘sample name’, ‘experiment’, ‘assay name’, and assay channels
(fluorescence dyes Fam or Vic/hex. NOTE: eva green is read on the same channel as
6-fam.).
6. Click ‘Experiment’ and a pull down menu with choices for experiment types will
be displayed. Choose ‘abs quant’. Select wells (highlight wells) and click apply or
press enter.
7. With the same wells highlighted, Select assay channels as unknown channel 1
(6FAM/ Evagreen). Click apply or press enter.
8. Name the assay – For ddTRAP I usually put ddTRAP and indicate the extension
time used (i.e., ddTRAP 40min ext) and press apply.
9. Name the samples.
10. Click run.
11. A screen will prompt you to save the template. Name your plate with a logical
name (record this in your lab note book or experiment log in excel etc.). Click save.
12. A screen will prompt you to pick the dye types (Fam/Vic or Fam/Hex) for
ddTRAP pick FAM/VIC.
13. A screen will prompt you to pick how the assay should be read: either rows or
columns. This is up to you but I typically read in columns. The order does not
matter.
14. Next the machine does a self-check to ensure the plate is loaded correctly and
that there are enough reagents to read the plate, if there are not enough reagents it
will prompt you to add more or abort the plate.
15. Wait until the first well is read. Each well takes about 90sec. 8*90sec = 12min
per column or 144 min per 12 column plate (give or take a few minutes).
5. Data analysis – Click the ‘analysis’ tab.
1. The data generated from the droplet reader is given is several formats. The most
informative data is the concentration of molecules per microliter (given under the
concentration header in the table view, this is also the number that will be displayed
in the well identification box). This number should be multiplied by 20L to give
the total concentration of molecules in the sample. Then to derive the molecules of
telomerase per cell, total molecules are divided by the number of cells input into the
reactions. See example data below.
Figure 2. ddTRAP heat map data output
from ddPCR. HeLa cell lysates
representing 2500 (100 cells, A01), 250
(10 cells, B01) and 25 (1 cells, C01) and
lysis buffer control (D01). The pink line
is the threshold line that separates
positive and negative droplets.
Adjusting this line in a sample with good
separation and little ‘rain’ (i.e., droplets
between the majority of positive and
negative droplets) will not effect the
measured concentration.
2. Data types –
A. CSV file. This file is likely the most useful for most applications. This file can be
exported and then converted into excel formatting and analyzed. This file contains
all the pertinent information including sample IDs, assay type, assay channel, well
location on plate, concentration per microliter, total number of droplets, number of
positive droplets, number of negative droplets, and confidence intervals for Poisson
distribution statistics of each measured samples. The way positive droplets are
converted into molecule counts is based upon a proprietary algorithm developed by
Biorad based on Poisson’s distribution. Essentially based upon the ratio of positive
droplets to total droplets the concentration of molecules per microliter is derived.
With sample partitioning this takes a logarithmic situation (PCR) and makes it linear
and digital (0 or negative or 1 and positive). Most assays tested are found to be
accurate over 5 orders of magnitude. The ideal number of molecules input into any
ddPCR assay is between 20 and 100,000 (for 20k droplets 20 molecules would give
a concentration of 1 molecule per microliter and 100,000 molecules would give a
concentration of 5000 molecules per microliter). Beyond this range of molecule
number the Poisson distribution error rate increases and the confidence intervals of
each measure will increase, thus reducing the confidence you can have in the
accuracy of your measurements. Thus for HeLa cells if 100 molecules of
telomerase are present per cell, then 100*50cells would give 5000 measured
molecules which is right in the correct dilution range for ddPCR, which
matches our empirical data that 50cell input gives the most reproducible
results. The confidence intervals for each sample gives the variability of the
measured number of molecules within each sample. To determine variability of a
series of replicates you would need to derive your own standard deviation and
means to calculate variability and confidence intervals.
B. Heat maps – On the QX200 data can be displayed as either a non-descript colors
or a heat map based upon droplet density at particular fluorescent amplitudes. I
think heat maps are the preferred way to visually display the raw data because it
gives you an idea of how many droplets are present in each population (positive or
negative) of droplets that directly corresponds to the molecule concentrations. You
can further adjust the display of the heat map by altering the axis of the graph by
clicking the options tap above the heat map and then altering the y-axis coordinates.
This helps when you have very high positive droplets but your population is
clustered at a lower fluorescence amplitude.
C. Events graphs- This is a bar graph that can used to depict the number of positive,
negative and total events recorded in a sample or series of samples. This graph will
show the consistency of total number of droplets if that is a concern for a certain
group of samples. Could be useful for a reviewer to see that event number was
consistent between samples with different concentrations.
D. Concentration plots- This is a plot of the Poisson concentration by well position
and includes the Poisson confidence interval of each sample. This shows well by
well assay variability.
Analyzing TRAP data –
Now that you know the types of data that ddPCR gives you we can analyzed TRAP
data. First, how final cell numbers are calculated:
If 50,000 cells were harvested, lysed in 40L, 1L of this lysate added to a 50L
telomerase extension and then 2L of the extension added to a 20L PCR reaction
we would have 50 cell equivalents of data as follows:
50,000/40 = 1250 cells/L lysate *1L/50L = 25 cells/L extension reaction *2L
into 20L ddPCR.
Since the concentration readout of ddPCR is given in molecules per microliter,
multiplying that number by 20L gives us the total concentration of active
telomerase molecules assayed from a given lysate.
For instance if the concentration from ddPCR was 10molecules/L, one would
multiple by 20L to get the total number of molecules (200 in this example). Then
one would divide by the number of cells input into the PCR reaction to derive the
molecules of telomerase per cells or 200/50= 4 molecules of active telomerase per
cell. The numbers used in this example do not reflect actual biology and are for
illustrative purposes only.