Ludlow
SOP Shay/Wright lab
1/27/13
Droplet Digital TRAP
Materials
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. 10M TS primer (HPLC purified)
6. 10M ACX primer (HPLC purified)
7. NP-40 lysis buffer (RNase/DNase-free): 10 mMTris-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 mMNaCl; 5 mM -mercaptoethanol; 0.1 mM AEBSF (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride)
8. 10 TRAP buffer (RNase/DNase-free): 200 mMTris-HCl, pH 8.3; 15 mM MgCl2; 630 mMKCl; 0.5% Tween 20; 10 mM EGTA
9. twin-tec 96 well plate – eppendorf951020362 (Fisher)
10. Easily pierced foil– less than 42m (Thermo ScientificAB0757).
11. LTS XLT Rainin pipet tips
12. Droplet generator cartridges (DG8), Biorad186-3008
13. Droplet generator oil, biorad186-3005
14. Droplet cartridge gaskets (DG8), biorad 186-3009
15. QX200 evagreenddPCRsupermix v2.0 (10028083 – for reference only not for sale).
16. 8 channel pipet, Rainin LTS lite (1-20L, and 20-200L).
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 40L = 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 50L. Prepare a master mix containing 1x TRAP buffer, 1x dNTP mix (50x = 2.5mM thus 0.05mM = 1x), 0.2M TS primer per sample, 20g*mL-1 BSA, and dH2O to 49L per sample. Add 1L of cell lysate to 49L of extension master mix. If cells lysed at 1250cells/uL then 1250cells/50L = 25 cells/microliter final concentration of extension. See example below:
Reagent / Volume (L) for 1x / Volume for 10x / Volume for 20x / Volume for 30x10x TRAP buffer / 5 / 50 / 100 / 150
2.5mM dNTP / 1 / 10 / 20 / 30
50mg/mL BSA / 0.4 / 4 / 8 / 12
10M TS / 1 / 10 / 20 / 30
1250 cells / L Lysate / 1 / - / - / -
dH2O / 41.6 / 416 / 832 / 1248
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.
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 EvagreenddPCR 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 22L 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 hotstarttaq 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 / Volume (L) 1x / Volume (L) 10x / Volume (L) 20x2x EvagreenddPCRsupermix (v2.0) / 11 / 110 / 220
10M TS / 0.11 / 1.1 / 2.2
10M ACX / 0.11 / 1.1 / 2.2
25 cells/L extension product / 2 / -
dH2O / 8.8 / 88 / 176
2. Set up droplet generation (DG) cartridge by first loading 20L into the sample well in the cartridge.
3. Load 70L 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 11L ddPCRsupermix with 11L 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 ~42L 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 (21m) 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 ~3hrs on the droplet reader.
2. Load the 96well plate into the plate holder matching well ‘A1’ with the ‘un-notched’ 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 20L 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.
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 40L, 1L of this lysate added to a 50L telomerase extension and then 2L of the extension added to a 20L PCR reaction we would have 50 cell equivalents of data as follows:
50,000/40 = 1250 cells/L lysate *1L/50L = 25 cells/L extension reaction *2L into 20L ddPCR.
Since the concentration readout of ddPCR is given in molecules per microliter, multiplying that number by 20L 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 20L 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.