1 Introduction

Cerebrospinal fluid (CSF) permeates brain tissue and directly reflects the biochemical processes that occur in the brain, assisted by the limitation that the blood brain barrier imposes to the free flow of molecules from brain to other compartments and the opposite way. Consequently, CSF is considered the best fluid to measure and identify new biomarkers for brain diseases [1]. At present, the diagnostic use of CSF biomarkers, although recommended for preclinical diagnoses of neurodegenerative diseases [2], still requires further research, validation, and standardization [3, 4]. Currently, the diagnosis of the different neurocognitive disease subtypes without biomarker evidence is difficult because many of these diseases exhibit similar clinical symptoms of dementia. Moreover, the majority of these diseases have a long preclinical phase that may last decades. Preclinical diagnosis relies on molecular biomarkers that are hypothesized to be linked with the underlying pathophysiology of the disease. However, the molecular mechanisms that underlie the majority of neurocognitive and neuropsychiatric diseases are still not well known, which hampers not only disease diagnosis but also the discovery of drugs capable of modifying the course of these diseases.

The pathophysiological biomarkers in the CSF currently used for preclinical diagnosis of neurodegenerative diseases are proteins which include amyloid beta 1-42 (Ab1-42), total levels of tau (t-tau), phosphorylated tau (p-tau), alpha-synuclein, and 14-3-3 proteins. A major difficulty for the diagnostic use of these biomarker proteins is that in body fluids some of them are found at very low concentrations, which fall near the inferior limit of the analytical sensitivity range of currently available protein detection techniques [5]. In addition, a major challenge in protein assays of body fluids in clinical analyses is the inability to establish absolute values that are reproducible and comparable between laboratories, mainly because protein quantification is based on antibody immunoassays that need to be referenced to an external standard and quantification requires a comparison with a standard calibration curve that is generally different amongst laboratories. An additional drawback is that the antibodies used for protein analysis may change significantly between different lots; for example, the epitopes recognized by the antibodies are generally unknown, the target epitopes differ markedly amongst polyclonal antibodies, and when monoclonal antibodies are used, the quality between different lots may vary. Another general source of variability for protein quantification in bodily fluids is the degree of protein integrity due to sample extraction, handling and long term storage. All these factors introduce a significant amount of variability in protein quantification assays in CSF or other bodily fluids that lessens reproducibility between laboratories.

To overcome the technical difficulties associated with protein quantification in CSF samples, we decided to shift the focus from proteins to deoxyribonucleic acid (DNA) because the latter may be less susceptible to degradation due to sample manipulation or long term storage. We opted to detect mitochondrial DNA (mtDNA) because we hypothesized that the amount of mtDNA in CSF could be an index of brain injury or metabolism. Based on this hypothesis, we found that a drop in mtDNA content in CSF is a biomarker for preclinical Alzheimer’s disease [6].

There are several advantages of measuring mtDNA over proteins as a biomarker in CSF. In theory, the circular nature of intact mtDNA should make it more resistant to potential degradation by endonucleases, which are likely present in CSF and other body fluids. Another advantage of mtDNA over protein in diagnostic assays is that the former is amenable to detection and quantification with PCR amplification techniques, which allow higher analytical sensitivity and specificity than immunoassay procedures. Indeed, the launch of digital PCR has made possible the precise detection and absolute quantification of nucleic acid content at a single-molecule resolution of target sequence [7, 8]. This is achieved by performing an end-point PCR reaction in a large number of sample partitions and applying Poisson analysis to the fraction of positive partitions for quantification. The ability to perform absolute quantification offered by digital PCR, avoiding the use of external reference standards, markedly improves repeatability and reproducibility of biomarker measurements. In addition, one of the advantages of the digital PCR protocol for quantifying the amount of mtDNA in CSF described here is that it does not require previous extraction or processing of the CSF sample, because we found that the presence of inhibitory molecules in CSF does not significantly influence end-point digital PCR quantification (Fig. 1c, d). Another important benefit of digital PCR is analytical sensitivity because it allows higher precision measurements than qPCR at low concentrations of the target sequence.

Fig. 1
figure 1

Analysis of cell-free mtDNA directly in unpurified CSF using hydrolysis probes or EvaGreen Fluorescent DNA-binding dye: Comparison between ddPCR and qPCR . Assay conditions were studied in a human CSF sample pool from several subjects. (a and b) Temperature gradient optimization assays were performed in a ddPCR reaction amplifying the 85 base pairs mtDNA amplicon (mtDNA-85) using a hydrolysis probe (a) or the EvaGreen fluorescent DNA binding dye (b). The maximum separation between positive (upper) and negative (lower) droplets for EvaGreen is reached at 66 °C and is constant between 50 and 66 °C, whereas for the hydrolysis probe assay, maximum separation is reached at approximately 60 °C and remains constant down to 50 °C. We choose 60 °C for maximum stringency in both assays, either in duplex or singleplex forms. In nontemplate controls (NTC) there is a nonsignificant number of positive (upper) droplets. (c, d) The influence of CSF volume in the PCR reaction was studied in hydrolysis probe (c) and EvaGreen fluorescent dye (d) assays using either ddPCR (square dots, continuous line) or qPCR (triangular dots, discontinuous line). In contrast to qPCR, addition of up to 6 μl of CSF to the PCR reaction did not inhibit ddPCR assay. (e) Accuracy and linearity of ddPCR reaction to directly measure mtDNA in CSF were assayed by adding different known concentrations of purified BAX-103 amplicons to a pooled CSF sample. Detection of mtDNA directly in CSF up to 3000 copies/μl fits to a linear model (r2 = 0.999). The detection limit under these conditions is less than 1 copy of target per microliter of CSF with a signal above the 95% confidence interval of average noise in nontemplate controls. (f) Representative image of an agarose gel showing a DNA ladder 75–20,000 base pairs (bp) and the Bax-103 (Lane 1) and mtDNA-85 (Lane 2) amplicons obtained in a CSF sample

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Droplet digital PCR (ddPCR) incorporates all the advantages of digital PCR by partitioning the reaction in oil emulsion droplets and analyzing the fraction of droplets that exhibit positive end-point PCR amplification [9, 10]. Here we describe a protocol to measure by ddPCR the concentration of cell-free circulating mtDNA directly in unpurified CSF using either hydrolysis probe or fluorescent DNA-binding dye methods with primers that produce an amplicon of 85 base pairs (mtDNA-85). A common problem of CSF samples is that some of them may be contaminated with cells that provide an erroneous measurement of cell-free mtDNA. To circumvent this problem, the hydrolysis probe protocol includes the simultaneous measurement of cell-free mtDNA and a nuclear gene in a multiplex ddPCR assay using the mtDNA-85 primers together with primers that amplify an amplicon of 103 base pairs (BAX-103) corresponding to the apoptosis regulator BAX isoform alpha. Alternatively, other single copy nuclear genes can be used for this purpose. The presence of at least one copy of the nuclear gene per microliter of CSF indicates that the sample is contaminated with genomic DNA and is excluded from analysis.

Characterization of the ddPCR reaction amplifying mtDNA in CSF with our protocol shows that the optimum temperature to reach the maximum separation between positive and negative droplets is 60 °C for both the hydrolysis probe (Fig. 1a) and the fluorescent dye (Fig. 1b). The optimal temperature for amplification of the BAX-103 amplicon is also 60 °C (data not shown) which allows the multiplex assay of this nuclear gene and mtDNA with hydrolysis probes. The volume of unpurified CSF sample that can be added to the ddPCR reaction without inhibition of the amplification due to factors present in CSF can be up to 6 μl in a total reaction volume of 20 μl. In contrast, this volume of CSF causes significant inhibition (approximately 25%) in qPCR reactions (Fig. 1c, d). In the ddPCR protocol we choose to use 4.5 μl of CSF because we found that this volume provides the highest signal with the lowest possibility of amplification inhibition. To check the possibility that, when individual CSF samples contain a high concentration of potential PCR inhibitors, this volume of CSF might inhibit the reaction, we measured CSF samples from subjects in an advanced stage of a rapid neurodegenerative disease that contain a very high content of neuronal death biomarkers such as tau (Fig. 2d, e). Despite a difference in approximately two orders of magnitude in the concentration of tau and possibly of many other proteins, we found no significant differences in the ddPCR reaction profile between these samples and the corresponding control samples. The absence of inhibition in the ddPCR is exemplified by a well-defined separation between the populations of positive and negative droplets in both low and high tau protein samples. In addition, the mean absolute values of FAM fluorescence of positive droplets are similar in low and high tau samples, showing that the PCR amplification reaches a similar endpoint in both reactions and that there are no inhibitory factors that differentially alter PCR in the samples (Fig. 2d, e).

Fig. 2
figure 2

Representative examples of results from ddPCR analyses of various CSF samples containing different levels of mtDNA or potential PCR inhibitors. The mtDNA content was measured in 4.5 μl of CSF sample, using the mtDNA-85 mix and the ddPCR procedure for hydrolysis probes. (a and b) Representative one-dimensional scatter plots of three replicates from a CSF sample containing a low concentration of mtDNA (a) and a different CSF sample containing a high concentration of mtDNA (b). Good repeatability is observed between replicates. The amount of mtDNA concentration does not influence the well-defined separation between positive (upper) and negative (lower) droplets. In addition, the proportion of positive/negative droplets is equivalent among replicates. The line drawn at 4000 fluorescence units represents the threshold used to define positive and negative droplets. (c) Histogram of total positive and negative partitions from assays shown in a and b. Threshold is determined by choosing a point between the two droplet partitions, for example, adding the mean fluorescence value of negative droplets plus the mean fluorescence value of positive droplets and dividing the result by 2. (d, e) Representative one-dimensional scatter plots of three replicates from a CSF sample containing a low concentration of tau protein (d) and a different CSF sample containing a high concentration of tau protein (e). In addition to different levels of tau, these two samples may contain also different amounts of other potential PCR inhibitors. Both samples exhibit a similar concentration of mtDNA and similar fluorescence values of positive and negative droplet populations, showing the resistance of ddPCR to the presence of potential PCR inhibitors

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The dynamic range, linearity, accuracy, and analytical sensitivity of our assay to measure the content of mtDNA and BAX in CSF were assessed by adding different known concentrations of the purified PCR amplicons to a sample of pooled CSF. Measurement of mtDNA or BAX in a sample volume of 4.5 μl of CSF is linear up to 3000 copies/μl of CSF, indicating that in the conditions of our ddPCR reaction there is no influence of the inhibitory factors present in the CSF on the multiplex detection of mtDNA and BAX as exemplified by the measurement of added copies of BAX into a pooled CSF sample (Fig. 1e). The limit of detection of our ddPCR reaction was close to the theoretical limit of three copies of target per reaction, corresponding to less than one copy of target per microliter of CSF with a signal above the 95% confidence interval of average noise in nontemplate controls. Agarose gel electrophoresis analyses confirmed that the ddPCR reaction conditions used in our experiments produced single amplicons of the corresponding size, 103 and 85 base pairs for Bax-103 and mtDNA-85 primer combinations, respectively (Fig. 1f).

2 Materials

2.1 Droplet Digital PCR Using Hydrolysis Probes

  1. 1.

    Forward and reverse primers and hydrolysis probe targeting mtDNA (see Note 1 ). Forward: mtDNA-85F, (5′-CTCACTCCTTGGCGCCTGCC-3′); reverse: mtDNA-85R (5′-GGCGGTTGAGGCGTCTGGTG-3′); hydrolysis probe: FAM-mtDNA-85P (6-carboxyfluorescein (FAM)-5′-CCTCCAAATCACCACAGGACTATTCCTAGCCATGCA-3′-Black Hole Quencher-1(BHQ-1)).

  2. 2.

    Forward and reverse primers and hydrolysis probe targeting a single copy nuclear gene, e.g., BAX. Forward: BAX-103F, (5′-TTCATCCAGGATCGAGCAGG-3′); reverse: BAX-103R, (5′-TGAGACACTCGCTCAGCTTC-3′) and hydrolysis probe: HEX-BAX-103P, (6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX)-5′-CCCGAGCTGGCCCTGGACCCGGT-3′-BHQ1). (see Notes 2 and 3 ).

  3. 3.

    ddPCR Supermix for Probes (No-dUTP) (catalog number 186-3023) (Bio-Rad, Hercules, CA, USA).

  4. 4.

    ddPCR Droplet Generation Oil for probes (catalog number 186-3005) (Bio-Rad).

2.2 Droplet Digital PCR Using Fluorescent DNA-Binding Dyes

  1. 1.

    Forward and reverse primers targeting mtDNA (see Note 1 ). Forward: mtDNA-85F, (5′-CTCACTCCTTGGCGCCTGCC-3′); reverse: mtDNA-85R, (5′-GGCGGTTGAGGCGTCTGGTG-3′).

  2. 2.

    Forward and reverse primers targeting a single copy nuclear gene, e.g., BAX. Forward: BAX-103F, (5′-TTCATCCAGGATCGAGCAGG-3′); reverse, BAX-103R (5′-TGAGACACTCGCTCAGCTTC-3′).

  3. 3.

    QX200™ ddPCR EvaGreen Supermix (catalog number 186-4033) (Bio-Rad). (see Note 3 )

  4. 4.

    QX200™ Droplet Generation Oil for EvaGreen (catalog number 186-4005) (Bio-Rad).

2.3 Common Materials to Assay Cerebrospinal Fluid Using ddPCR

  1. 1.

    Cerebrospinal fluid sample (see Note 4 ).

  2. 2.

    Calibrated micropipettes and low retention, presterilized, aerosol barrier filter pipette tips, (Biotix, San Diego, CA, USA)(see Note 5 ).

  3. 3.

    Polypropylene PCR tubes, 0.2 ml.

  4. 4.

    Vortex mixer.

  5. 5.

    Microcentrifuge (5000 × g).

  6. 6.

    DG8™ Cartridges for QX200™/QX100™ Droplet Generator (catalog number 186-4008) (Bio-Rad).

  7. 7.

    DG8™ Cartridge Holder (catalog number 186-3051) (Bio-Rad).

  8. 8.

    DG8™ Gaskets for QX200™/QX100™ Droplet Generator (catalog number 186-3009) (Bio-Rad).

  9. 9.

    Electronic multichannel pipette with wide bore tips (see Note 6 ).

  10. 10.

    Eppendorf twin.tec® 96-Well PCR plates, semiskirted (catalog number 0030 128.XXX) (Eppendorf).

  11. 11.

    PX1™ PCR Plate Sealer (catalog number 186-4000) (Bio-Rad).

  12. 12.

    Pierceable Foil Heat Seal (catalog number 181-4040) (Bio-Rad) (see Note 7 ).

  13. 13.

    C1000 Touch™ Thermal Cycler with 96-Deep Well Reaction Module (catalog number 185-1197) (Bio-Rad).

  14. 14.

    ddPCR Droplet Reader Oil (catalog number 186-3004) (Bio-Rad).

  15. 15.

    QX200™ Droplet Digital™ PCR System (catalog number 186-4001) (Bio-Rad).

  16. 16.

    QuantaSoft™ Software (catalog number 186-4011) (Bio-Rad).

3 Methods

3.1 Sample Acquisition and Processing

CSF should be collected and processed following standard operating procedures after informed consent of the patient. In our studies, CSF was collected from subjects recruited at the Alzheimer’s disease and other cognitive disorders unit of the Hospital Clinic of Barcelona. The CSF samples were obtained with informed consent at the Alzheimer’s disease and other cognitive disorders Unit of the Neurology Service, following the procedure approved by the ethics committee of the Hospital Clinic of Barcelona. CSF was obtained by lumbar puncture between 9 a.m. and 12 p.m. A small sample of the first CSF volume was reserved for later measurement of cell contamination and this was followed by collection of 10 ml of CSF. The whole volume of CSF was centrifuged (2000 × g/4 °C/10 min), aliquoted after centrifugation in 500 μl polypropylene tubes and stored at −80 °C within two hours of the beginning of the collection. The mtDNA assay was performed in unpurified CSF that underwent only one thaw. It is convenient to avoid using CSF samples that have undergone repeated freeze–thaw cycles. However, we find that up to three freeze–thaw cycles do not significantly modify the measured concentration of mtDNA by ddPCR.

3.2 Digital PCR Reaction Setup and Amplification

Bring all reagents and samples to room temperature before use. Centrifuge briefly, mix thoroughly by vortexing and centrifuge briefly again to properly mix and collect the components to the bottom of the tubes.

  1. 1.

    Prepare a master mix in a polypropylene microcentrifuge tube containing ddPCR Supermix, oligonucleotide mix, and double-distilled nuclease-free water in a total volume sufficient to obtain 15.5 μl per assay as shown in Table 1 (see Note 8 ). It is essential to include nontemplate control assays, with master mix but adding nuclease-free dH2O instead of CSF sample (see Note 9 ). In addition, for assay characterization, it is necessary to include positive control samples containing known amounts of nuclear DNA or mitochondrial DNA target amplicons cloned in vectors or purified from PCR reactions (see Note 10 ). The process of assay characterization includes assessment of accuracy, sensitivity, setting the threshold for positive droplets and the level of false positive droplets for mtDNA and nuclear DNA respectively.

    1. (a)

      The ddPCR assay using hydrolysis probe is designed as a multiplex assay to monitor simultaneously mtDNA and BAX in the same reaction. The composition of the master mix for ddPCR using hydrolysis probes is as shown in Table 1 (see Note 11 ):

    2. (b)

      The ddPCR assay of mtDNA and the nuclear gene BAX using fluorescent DNA binding dyes is performed in separate reactions. The composition of the master mix for ddPCR using EvaGreen fluorescent dye is as shown in Table 2 Master mix for ddPCR using EvaGreen fluorescent dye (see Note 8 ).

  2. 2.

    Distribute 15.5 μl of the master mix with a 20 μl pipette to 0.2 ml Polypropylene PCR tubes (see Note 8 ).

  3. 3.

    Add 4.5 μl of unpurified CSF sample to the tube (see Note 8 ).

  4. 4.

    Mix well by vortexing and centrifuge briefly to collect the mix to the bottom of the tube and remove bubbles. The Supermix is viscous and insufficient mixing will lead to less reproducible results.

  5. 5.

    Place the DG8™ Cartridge for Droplet Generator into the DG8™ Cartridge Holder.

  6. 6.

    Transfer each 20 μl of reaction mixture to one of the eight sample wells in the middle of the DG8™ Cartridge (see Note 8 ).

  7. 7.

    Add 70 μl of either ddPCR Droplet Generation Oil for probes (for the probe hydrolysis method) or QX200™ Droplet Generation Oil for EvaGreen (for the fluorescent DNA-binding dye method) to the oil wells of the DG8™ Cartridge adjacent to the samples (see Note 12 ).

  8. 8.

    Cover the cartridge with a DG8™ Gasket for QX200™/QX100™ Droplet Generator.

  9. 9.

    Place the covered cartridge into the QX100/QX200™ Digital Droplet Generator to generate the droplets (see Note 13 ).

  10. 10.

    Transfer 40 μl of the emulsified sample to an Eppendorf twin.tec® 96-Well semiskirted PCR plate with an electronic multichannel pipette set on the lowest aspirating and dispensing speed using wide bore tips. Make sure to keep the pipet at a 15°–30° angle when aspirating the emulsified sample to avoid damaging the droplets.

  11. 11.

    Cover the 96-well PCR plate with pierceable foil heat seal and place it into PX1™ plate sealer. Seal at 170 °C for 4 s.

  12. 12.

    Place the Eppendorf twin.tec® semiskirted 96-Well PCR plate into a PCR thermal cycler that has the ability to adjust a ramp temperature and that is compatible with deep wells. Set ramp rate to 2 °C per second for all steps.

  13. 13.

    Use the following PCR profiles (see Note 14 ):

    1. (a)

      For hydrolysis probes:

      • 95 °C: 10 min.

      • 40 cycles:

        • 94 °C: 30 s.

        • 60 °C: 1 min.

      • 98 °C: 10 min.

      • 4 °C: infinite.

    2. (b)

      For fluorescent dyes (see Note 15 ):

      • 95 °C: 5 min.

      • 40 cycles:

        • 95 °C: 30 s.

        • 60 °C: 1 min.

      • 4 °C: 5 min.

      • 90 °C: 5 min.

      • 4 °C: infinite.

  14. 14.

    After PCR the plate can be left in the thermal cycler overnight or stored for a few days at 4 °C before droplet reading.

Table 1 Master mix for ddPCR using hydrolysis probes (see Note 8 )
Full size table
Table 2 Master mix for ddPCR using EvaGreen fluorescent dye (see Note 8 )
Full size table

3.3 Digital Data Acquisition

  1. 1.

    Power on the QX200™ Droplet Digital™ PCR Droplet Reader and let it warm up for at least 30 min, making certain that there is enough ddPCR Droplet Reader Oil in the reader compartment. Prime the system if the instrument has been unused for longer than a week.

  2. 2.

    Place the 96-well PCR plate to the QX200™ Droplet Digital™ PCR Droplet Reader.

  3. 3.

    Introduce the appropriate settings in the PCR Droplet reader software indicating the PCR supermix used and the fluorescence acquisition channels (see Note 15 ).

3.4 Data Analysis

  1. 1.

    After reading all samples, open the file with the appropriate QuantaSoft software and in the Experiments window select Absolute Quantification method (ABS) and then select all samples for data analysis.

  2. 2.

    Click on the Analyze tab and then the multiwell thresholding icon. Choose a point between the two droplet populations shown in the fluorescence histogram (Fig. 2c) to identify a threshold value that reliably separates positive and negative droplets. For example, add the mean fluorescence value of negative droplets plus the mean fluorescence value of positive droplets and divide the result by 2. Enter the resulting value into the Set Threshold window (see Note 16 )

  3. 3.

    Determine target concentrations by analyzing the proportion of negative versus positive droplets using the Absolute Quantification method (see Note 17 ). Replicates that do not exhibit equivalent mean fluorescence values of positive and negative droplets between them indicate a droplet generation failure and should be discarded. When all replicates of one sample exhibit equivalent mean fluorescence that is significantly lower than the exhibited by the replicates of another sample, this indicates inhibition of the PCR and values cannot be compared.

  4. 4.

    Obtain the total number of copies per 20 μl of reaction for each target and divide by the volume (e.g., 4.5 μl) of CSF introduced into the reaction to obtain copies per microliter of CSF.

4 Notes

  1. 1.

    These primers target an mtDNA sequence that is not present in the nuclear genome as a nuclear mtDNA (Numt). When designing primers targeting other mtDNA sequences it is important to check that these sequences do not have a corresponding Numt.

  2. 2.

    The reporter fluorophores used to label the hydrolysis probes can be FAM combined interchangeably with either HEX or VIC in any of the probes. We chose to use FAM for mtDNA and HEX for BAX, but it could also be the opposite. It is likely that in the near future there will be multichannel digital PCR platforms that will allow more fluorophore combinations for multitarget detection. We use the dark quencher BHQ-1 because it is capable of quenching both FAM and HEX with high efficiency. We found that quenchers such as TAMRA do not completely quench probe fluorescence and do not permit a clear separation between positive and negative droplets.

  3. 3.

    The oligomix with fluorophores or EvaGreen supermix should not be exposed to light for prolonged periods of time. We cover with aluminum foil all tubes that contain fluorophores.

  4. 4.

    After centrifugation of the whole volume of CSF obtained by lumbar puncture, the unpurified CSF should be placed in sterile siliconized polypropylene microcentrifuge tubes to avoid mtDNA absorption to the tube, and stored at −80 °C in small aliquots (e.g., 500 μl) to avoid unnecessary transfer to different tubes or freezing–thawing cycles.

  5. 5.

    The use of low retention pipette tips is essential to obtain appropriate accuracy and repeatability of mtDNA measurements in CSF. Low retention tips are designed to sustain minimal sample loss from viscous liquids such as CSF. Regular calibration of pipettes and correct pipetting techniques with slow aspiration/delivery rates is crucial to get low coefficients of variation for repeatability and reproducibility.

  6. 6.

    Wide bore tips with an orifice size wider that 1.5 mm are convenient to prevent mechanical damage to the droplets when transferring from the DG8 cartridge to the PCR plate.

  7. 7.

    This foil seal cannot be replaced by another material because it may damage the ddPCR droplet reader.

  8. 8.

    We find that it is best to prepare the master mix for all the planned reaction tubes in an excess volume of 10% and distribute to each tube 17.05 μl of master mix. To this volume, we also add a 10% excess of CSF sample (4.95 μl). This is because in Subheading 3.2.6 of the protocol it is necessary to transfer without bubbles exactly 20 μl of this mixture to the cartridge well, and we found that if the initial volume is exactly 20 μl it is very difficult not to draw bubbles in the pipette during the transfer.

  9. 9.

    We find it is important that each ddPCR plate contains several nontemplate control assays (NTCs) that have master mix and nuclease-free dH2O instead of CSF sample. NTCs allow identification of possible contamination of analysis materials and reagents. It is essential to perform all procedures with sterile gloves using material specially reserved for PCR in laboratory areas only dedicated to the setup of PCR reactions.

  10. 10.

    Care should be taken to avoid contamination of equipment and samples with DNA standards that may contain significantly higher concentrations than the samples.

  11. 11.

    The reaction mixture contains a different concentration of FAM versus HEX labelled probe. The reason is that we found that there is a marked bleed-through cross-emission of FAM into HEX channel. One way to reduce such fluorophore cross-emission is by lowering the concentration of FAM labelled probe. The concentration ratio of FAM/HEX in our reaction conditions is optimized for our particular probes and if using other probes or quenchers we recommend finding again the optimal conditions.

  12. 12.

    It is important to add the oil after the reaction mixture because if added before, it will block the microfluidic channels.

  13. 13.

    We find that it is important to inspect the cartridge after droplet generation to ensure that droplets have been correctly formed. A simple way when using hydrolysis probes is to slowly rotate the cartridge in front of a light source and if droplets are formed there is a change in reflection of the light source every 60° rotation. If there are no droplets in one of the wells of the cartridge (either because of air bubbles or obstruction in the microfluidic channel) it is advisable to discard the whole cartridge. When using EvaGreen fluorescent dye it is more difficult to identify whether droplets have been correctly formed prior to the analysis.

  14. 14.

    The PCR temperature profiles described here are the optimal for the particular primer combinations of our reactions and our laboratory conditions. We recommend to follow the digital MIQE guidelines [11] and perform annealing temperature gradients to optimize the separation between positive and negative droplets for each primer combination and laboratory setting.

  15. 15.

    The total time to analyze a 96-well plate is approximately 7 h. This includes: 2.5 h for sample preparation and droplet generation: 2 h for thermocycling and 2.5 h for droplet reading.

  16. 16.

    We recommend setting the same threshold value for all the wells in the plate (Fig. 2). Some samples may show only one of the droplet partitions and the fluorescence histogram of those samples will have only one Gaussian curve, hampering the detection of a reliable threshold value to separate positive and negative partitions. Therefore, we find convenient to select all wells for analysis because then the histogram shows the amplitude of the total events in all samples, making unlikely the possibility that there is only one droplet population in all of them. We find that when the ddPCR assay is performed in the most optimal conditions, the threshold value does not significantly influence the result.

  17. 17.

    Only samples that exhibit a total of more than 12,000 droplets or events should be included in the final analysis. However, for most accurate measurements, setting the limit for sample inclusion to more than 15,000 droplets provides results with significantly lower coefficient of variation among replicates. Using this ddPCR method to measure mtDNA concentration in 4.5 μl of CSF we find that three sample replicates is sufficient to obtain precise values with repeatability levels equivalent to the sampling error expected for volumes of less than 10 μl.