six rnase - free , hplc - purified 5-phosphorylated mirna oligoribonucleotides were synthesized ( integrated dna technologies ) for the analytical portion of this study , corresponding to hsa - mir-16 ( 5-phospho - uagcagcacguaaauauuggcg - oh-3 ) , hsa - mir-141 ( 5-phospho - uaacacugucugguaaagaugg - oh-3 ) , hsa - mir-135b ( 5-phospho - uauggcuuuucauuccuauguga - oh-3 ) , hsa - mir-205 ( 5-phospho - uccuucauuccaccggagucug - oh-3 ) , hsa - mir-210 ( 5-phospho - cugugcgugugacagcggcuga - oh-3 ) and hsa - mir-375 ( 5-phospho - uuuguucguucggcucgcguga - oh-3 ) .
stock solutions of 10 m synthetic oligonucleotide in rnase - free / dnase - free water were prepared according to the concentrations and sample purity quoted by the manufacturer ( based on spectrophotometry analysis ) . to minimize uncertainty resulting from pipetting , all dilutions where the volumetric dilution factor exceeded 2 ( see supplementary table 1 , dilutions 14 )
were performed on an analytical balance , such that the predicted copies going into the standard curves could be accurately scaled using a gravimetric dilution factor .
all further dilutions for the standard curve ( supplementary table 1 , dilutions 511 ) were performed volumetrically .
approximately 55,000 copies per 20 l pcr reaction with nine intermediate twofold serial dilutions and additional no template controls ( ntcs ; zero copies ) were examined .
dilution series for each of the synthetic mirnas were made in either rnase / dnase - free h2o or in healthy human donor plasma rna solution to provide a constant background of endogenous mirnas .
our modified procedure used for isolating mirnas from plasma with mirneasy kits ( qiagen ) has been described previously . to ensure a homogenous plasma rna solution for use as diluent ,
multiple plasma rna extraction batches collected using this protocol were pooled and mixed by gentle pipetting , then divided into 60-l aliquots and stored at 80 c until use .
rt of input mirnas ( supplementary table 1 , dilutions 511 ) was conducted using reagents from the taqman mirna reverse transcription kit and 60 target - specific rt primers ( both from applied biosystems , inc . ) .
the kit contains the following components that , together with the stem - loop primers and rnase / dnase - free h2o , comprise what is forthwith referred to as 
rt mix ( numbers in parentheses are final volumes in a 10-l rt reaction containing 8 l rt mix ) : 10 rt buffer ( 1 l ) , rnase inhibitor ( 0.12 l ) , 100 mm each dntp ( 0.1 l ) , multiscribe reverse transcriptase ( 0.66 l ) , 60 rt primer ( 0.17 l ) , rnase / dnase - free h2o ( 5.95 l ) .
the volumetric ratio of rt mix to input mirna solution was 4:1 , and triplicate 10 l rt reactions ( supplementary fig .
1 ) for each point of the curves were taken from a corresponding well - mixed stock solution of a given concentration ( 34 l total volume ; 7 l input mirna ) . the reverse transcription thermal - cycling procedure for triplicate 10 l rts used holds at 16 c for 30 min , then 42 c for 30 min and 85 c for 5 min ( tetrad2 peltier thermal cycler ; bio - rad ) .
7 l of rt product for each concentration was thoroughly mixed by pipette resuspension with 133 l solutions containing master mix ( bio - rad , 70 l of 2 mix ) , the target - specific taqman real time pcr primer probe set ( applied biosystems , 2.33 l of 60 solution ) and rnase / dnase - free h2o ( 60.67 l ) . from this bulk solution ,
triplicate pcr reactions were carried out using both ddpcr ( 20 l pcr reaction ) and real - time pcr ( 5 l pcr reaction ) , such that both ddpcr and real - time pcr reactions had identical concentrations of synthetic mirna oligonucleotides .
real - time pcr experiments were performed on an applied biosystems viia 7 instrument with the following thermal - cycling procedure ; 95 c for 10 min , followed by 40 cycles of 95 c for 15 s and 60 c for 1 min ( 1.6 c / s ramp rate ) as specified in the taqman microrna assay protocol provided by the manufacturer ( applied biosystems ) . to provide the best possible analysis of the raw real - time pcr data for comparison to ddpcr , real - time pcr data collected in this manner were analyzed with the viia
7 instrument software v1.0 using three separate conventional approaches and compared side by side to deduce which method gave the lowest variability ( data not shown ) .
when using the first method , we examined the effect of using a universal baseline and threshold to aid data consistency across the study .
the baseline was set between 8 cycles and 20 cycles ( to base this on the background signal found in early cycles of amplification ) with a manual fluorescence threshold of 20,000 relative fluorescence units ( rfus ) with rox ( carboxy - x - rhodamine passive reference dye ) normalization disabled ( ddpcr master mix does not contain rox dye ) . in the second method , the threshold was manually set by the operator to exclude spurious noise and intersect the exponential amplification portion of the fluorescence curve as centrally as possible .
the third method used the viia 7 signal processing algorithm to automatically call baseline and threshold , with rox normalization disabled .
method 3 was found to give the lowest variability between pcrs , and therefore this method was used for analysis of all data derived from real - time pcr analysis of ddpcr reaction mixtures .
note that data derived from the standard real - time pcr protocol ( i.e. , used to analyze clinical specimens ) was analyzed using its corresponding data analysis procedure , which was identical to method 3 above with the exception of inclusion of rox normalization .
curves that did not show typical exponential amplification morphology were included in the presentation of the whole data ( fig .
2 ) but were excluded from subsequent summary operating characteristic and statistical analysis ( supplementary figs .
36 and supplementary tables 25 ) to provide the best possible estimate of real - time pcr performance .
undetermined cycle thresholds were arbitrarily set to 40 as a limiting estimate of the maximum possible abundance of target .
target abundance relative to the mean of highest concentration tested was determined by pfaffl analysis of the cycle - threshold ( ct ) data , using the empirically determined pcr efficiency for each mirna . in the case of the standard curves generated for the analysis of clinical specimens ,
limit of quantification ( loq ) was defined as the lowest concentration tested that remained above or equal to the lower limit of linear range of the assay ( lllr ) and above or equal to the limit of detection ( lod ) .
linear range was determined by runs - testing , removing successive dilution points until the p value was > 0.05 , indicating no significant deviation from linearity ( prism version 5.0c software ) .
lod was defined as < x > bi + ksbi , where < x > bi equals the mean of the no - template controls , sbi is s.d . of no - template controls , and k = 2.479 ( 99% confidence interval ) .
the ddpcr system , workflow , operating characteristics and analytical performance has been recently described in detail by some of the authors of this paper in two recent publications , but a brief summary of this technology follows . at the start of these studies ,
the instrument and reagents used were manufactured by quantalife , inc . , which was subsequently acquired by bio - rad , inc .
each 20 l pcr reaction ( see above ) was loaded into an 8-channel , single - use consumable droplet generation cartridge .
60 l of oil containing emulsion - stabilizing , biocompatible surfactant was loaded into adjacent oil wells , and the microfluidic chip was loaded into a beta - series prototype droplet generator ( dg ) .
the dg applies a vacuum to the outlet well creating a pressure difference across the cartridge that simultaneously partitions the sample present in each of the 8 wells into 20,000 monodisperse droplets of accurately known volume .
the resulting water - in - oil emulsions were pipette - transferred from the outlet well to a 96-well polypropylene plate ( eppendorf ) , sealed with foil and then amplified to endpoint using a tetrad2 peltier thermal cycler ( bio - rad ) and the cycling protocol : 95 c for 10 min then 40 cycles of 95 c for 15 s and 60 c for 1 min ( 2.5 c / s ramp rate ) with a final 10 min hold at 98 c . plates containing amplified droplets were loaded into an early - access , beta - version of the commercially available qx100 droplet reader ( bio - rad ) , which aspirates droplets from the 96-well plate , one well at a time , and streams them single - file ( 1,500 droplets / s ) past a two - color fam / vic ( fam and vic fluorescence dyes , life technologies corp . )
discrimination between droplets that did not contain target ( negatives ) and those that did ( positives ) was achieved by applying a global fluorescence amplitude threshold .
for all of the mirna assays , the global fluorescence threshold was set at 4,000 relative fluorescence units ( rfus ) regardless of the assay efficiency .
concentration estimates (  ) were based on the fraction of droplets where amplification has occurred ( p ) modeled as a poisson distribution ( equation ( 1 ) ) . because each droplet is an independent pcr reaction vessel of equal and defined volume , the droplets of technical replicates ( triplicate wells ) can be pooled to create a 
metawell. to compute total  95% confidence intervals about concentration estimates of metawells ( comprised of previously described poisson  95% cis and  real world error ; concentration differences between wells ) we applied the following meta - analysis techniques .
given k replicates with concentrations m1 , m2 ,  mk and poisson variances v1 , v2 ,  vk , respectively , we define the weight ( w ) of replicate i as reciprocal of its variance as let m be the weighted average of concentrations and consider the following random variable that measures fluctuation of concentrations around this weighted mean : this is the sum of squares of approximately standard normal random variables and can therefore be approximated as a chi - squared distribution .
the mean of the distribution is the number of degrees of freedom ( df = k  1 ) .
if t is less than df , we say that there is no additional real - world variance .
if t is more than df , then it suggests there is additional real - world variance r = t  df .
as t is based on standard normal variables , we scale back r to r in original units after applying an appropriate correction factor : we can add r to poisson variance to give total variance for each replicate .
we redefine the weight of each replicate as : from which we can compute the total uncertainty around the metawell as the final estimate m is recomputed as weighted average of concentrations with new weights . by setting r to 0
this meta - analysis is a statistically rigorous method to test the existence of real - world error in a group of replicate droplet digital pcr wells ( us patent application 20130017551 ) .
all of these data - analysis methods are implemented in the quantasoft ( 1.1.1.0 ) data analysis package , installed with the droplet reader .
human serum samples from individuals with metastatic prostate cancer and prostate - cancer  negative controls were collected after written informed consent was obtained .
all participants signed a university of washington human subjects committee approved informed consent form for a peripheral blood draw and the research was approved and supervised by the university of washington and fred hutchinson cancer research center institutional review boards .
prostate - cancer  negative donors were recruited among individuals undergoing screening for prostate cancer and found to be negative by digital - rectal examination ( dre ) and serum psa analysis .
individuals with metastatic prostate cancer were recruited among previously diagnosed patients undergoing treatment at the university of washington ( seattle ) .
a smaller cohort of clinical specimens corresponding to n = 20 advanced prostate cancer cases and n = 20 healthy controls was analyzed ( previously , n = 25 each group ) .
the patient specimens studied here were distinct from ( not a subset of ) those we studied previously . using the population - variance data from the previous study , we performed a power calculation and determined that n = 12 individuals per group would result in 81% power to detect a difference in means of fourfold ( alpha = 0.05 , two tails ) .
we chose to analyze specimens from n = 20 individuals per group , as this was above the minimum predicted by the power calculation , feasible and simplified the technical work relative to the previous study .
the experimenter was kept blinded to case status ( i.e. , cancer versus control ) . for comparing the performance of ddpcr to real - time pcr in the clinical specimens
synthetic mir-141 standard curves used in this portion of the study were made using ms2 carrier rna , which leads to improved circulating mirna precision in real - time pcr analyses and therefore was included to provide the best possible real - time pcr performance to compare with ddpcr .
ddpcr showed superior precision ( supplementary fig . 8) , consistent with our results displayed in figure 1a , b and comparable limits of quantification ( supplementary table 7 ) for mir-141 in the analyses of these standard curves .
owing to limited serum rna volume for the large number of replicates needed for this study , we diluted the initial serum rna specimens by a factor of 1.5 . to allow a direct comparison of performance between ddpcr ( which uses bio - rad ddpcr supermix for probes , 186 - 3010 ) and standard circulating mirna real - time pcr ( which uses abi taqman universal pcr mastermix no ung , 4326614 ) , differences in the reagent volumes used between the bio - rad ddpcr and the standard circulating mirna real - time pcr protocols needed to be accommodated to achieve the same concentration of cdna in each comparable reaction across the methods ( supplementary fig . 7 ) .
as a result of these necessary adjustments , the concentration of input cdna analyzed per reaction for both ddpcr and real - time pcr methods was ultimately about tenfold lower than that used previously . after reverse transcription , the cdnas corresponding to the sets of standard curves ( supplementary fig . 8) and the 40 clinical specimens ( fig .
2 ) were each split into six aliquots for subsequent pcr reactions ( see below ) .
three of these six cdna aliquots were added to abi master mix for subsequent real - time pcr , and the remaining three were added to bio - rad mastermix to conduct ddpcr and real - time pcr ( fig .
l ddpcr master mix for each sample was divided into three 20 l aliquots for analysis by ddpcr and three 5 l aliquots for analysis by real - time pcr .
the prepared 20 l standard real - time pcr master mix for each sample was divided into 3  5 l aliquots for analysis by real - time pcr ( fig . 2 and supplementary fig . 7 ) . for the analysis of clinical specimens ,
ddpcr triplicates were combined into a single metawell before poisson analysis and recovery correction by caenorhabditis elegans recovery - yield mirna spike - ins . for real - time pcr ,
the concentration of analyte was calculated by ct comparison to the standard curve , and results were presented as the mean of the pcr triplicates corrected by c. elegans spike - ins .
statistical significance was determined by mann - whitney analysis ( 95% confidence intervals , two tails ) and receiver operating characteristic ( roc ) analysis was performed using prism 5.0c software .
six rnase - free , hplc - purified 5-phosphorylated mirna oligoribonucleotides were synthesized ( integrated dna technologies ) for the analytical portion of this study , corresponding to hsa - mir-16 ( 5-phospho - uagcagcacguaaauauuggcg - oh-3 ) , hsa - mir-141 ( 5-phospho - uaacacugucugguaaagaugg - oh-3 ) , hsa - mir-135b ( 5-phospho - uauggcuuuucauuccuauguga - oh-3 ) , hsa - mir-205 ( 5-phospho - uccuucauuccaccggagucug - oh-3 ) , hsa - mir-210 ( 5-phospho - cugugcgugugacagcggcuga - oh-3 ) and hsa - mir-375 ( 5-phospho - uuuguucguucggcucgcguga - oh-3 ) .
stock solutions of 10 m synthetic oligonucleotide in rnase - free / dnase - free water were prepared according to the concentrations and sample purity quoted by the manufacturer ( based on spectrophotometry analysis ) . to minimize uncertainty resulting from pipetting , all dilutions where the volumetric dilution factor exceeded 2 ( see supplementary table 1 , dilutions 14 )
were performed on an analytical balance , such that the predicted copies going into the standard curves could be accurately scaled using a gravimetric dilution factor .
all further dilutions for the standard curve ( supplementary table 1 , dilutions 511 ) were performed volumetrically .
approximately 55,000 copies per 20 l pcr reaction with nine intermediate twofold serial dilutions and additional no template controls ( ntcs ; zero copies ) were examined .
dilution series for each of the synthetic mirnas were made in either rnase / dnase - free h2o or in healthy human donor plasma rna solution to provide a constant background of endogenous mirnas .
our modified procedure used for isolating mirnas from plasma with mirneasy kits ( qiagen ) has been described previously . to ensure a homogenous plasma rna solution for use as diluent ,
multiple plasma rna extraction batches collected using this protocol were pooled and mixed by gentle pipetting , then divided into 60-l aliquots and stored at 80 c until use .
rt of input mirnas ( supplementary table 1 , dilutions 511 ) was conducted using reagents from the taqman mirna reverse transcription kit and 60 target - specific rt primers ( both from applied biosystems , inc . ) .
the kit contains the following components that , together with the stem - loop primers and rnase / dnase - free h2o , comprise what is forthwith referred to as 
rt mix ( numbers in parentheses are final volumes in a 10-l rt reaction containing 8 l rt mix ) : 10 rt buffer ( 1 l ) , rnase inhibitor ( 0.12 l ) , 100 mm each dntp ( 0.1 l ) , multiscribe reverse transcriptase ( 0.66 l ) , 60 rt primer ( 0.17 l ) , rnase / dnase - free h2o ( 5.95 l ) .
the volumetric ratio of rt mix to input mirna solution was 4:1 , and triplicate 10 l rt reactions ( supplementary fig .
1 ) for each point of the curves were taken from a corresponding well - mixed stock solution of a given concentration ( 34 l total volume ; 7 l input mirna ) . the reverse transcription thermal - cycling procedure for triplicate 10 l rts used holds at 16 c for 30 min , then 42 c for 30 min and 85 c for 5 min ( tetrad2 peltier thermal cycler ; bio - rad ) .
7 l of rt product for each concentration was thoroughly mixed by pipette resuspension with 133 l solutions containing master mix ( bio - rad , 70 l of 2 mix ) , the target - specific taqman real time pcr primer probe set ( applied biosystems , 2.33 l of 60 solution ) and rnase / dnase - free h2o ( 60.67 l ) . from this bulk solution ,
triplicate pcr reactions were carried out using both ddpcr ( 20 l pcr reaction ) and real - time pcr ( 5 l pcr reaction ) , such that both ddpcr and real - time pcr reactions had identical concentrations of synthetic mirna oligonucleotides .
real - time pcr experiments were performed on an applied biosystems viia 7 instrument with the following thermal - cycling procedure ; 95 c for 10 min , followed by 40 cycles of 95 c for 15 s and 60 c for 1 min ( 1.6 c / s ramp rate ) as specified in the taqman microrna assay protocol provided by the manufacturer ( applied biosystems ) . to provide the best possible analysis of the raw real - time pcr data for comparison to ddpcr , real - time pcr data collected in this manner were analyzed with the viia 7 instrument software v1.0 using three separate conventional approaches and compared side by side to deduce which method gave the lowest variability ( data not shown ) .
when using the first method , we examined the effect of using a universal baseline and threshold to aid data consistency across the study .
the baseline was set between 8 cycles and 20 cycles ( to base this on the background signal found in early cycles of amplification ) with a manual fluorescence threshold of 20,000 relative fluorescence units ( rfus ) with rox ( carboxy - x - rhodamine passive reference dye ) normalization disabled ( ddpcr master mix does not contain rox dye ) . in the second method ,
the threshold was manually set by the operator to exclude spurious noise and intersect the exponential amplification portion of the fluorescence curve as centrally as possible .
the third method used the viia 7 signal processing algorithm to automatically call baseline and threshold , with rox normalization disabled .
method 3 was found to give the lowest variability between pcrs , and therefore this method was used for analysis of all data derived from real - time pcr analysis of ddpcr reaction mixtures .
note that data derived from the standard real - time pcr protocol ( i.e. , used to analyze clinical specimens ) was analyzed using its corresponding data analysis procedure , which was identical to method 3 above with the exception of inclusion of rox normalization .
curves that did not show typical exponential amplification morphology were included in the presentation of the whole data ( fig .
2 ) but were excluded from subsequent summary operating characteristic and statistical analysis ( supplementary figs .
36 and supplementary tables 25 ) to provide the best possible estimate of real - time pcr performance .
undetermined cycle thresholds were arbitrarily set to 40 as a limiting estimate of the maximum possible abundance of target .
target abundance relative to the mean of highest concentration tested was determined by pfaffl analysis of the cycle - threshold ( ct ) data , using the empirically determined pcr efficiency for each mirna . in the case of the standard curves generated for the analysis of clinical specimens ,
limit of quantification ( loq ) was defined as the lowest concentration tested that remained above or equal to the lower limit of linear range of the assay ( lllr ) and above or equal to the limit of detection ( lod ) .
linear range was determined by runs - testing , removing successive dilution points until the p value was > 0.05 , indicating no significant deviation from linearity ( prism version 5.0c software ) .
lod was defined as < x > bi + ksbi , where < x > bi equals the mean of the no - template controls , sbi is s.d . of no - template
the ddpcr system , workflow , operating characteristics and analytical performance has been recently described in detail by some of the authors of this paper in two recent publications , but a brief summary of this technology follows . at the start of these studies ,
the instrument and reagents used were manufactured by quantalife , inc . , which was subsequently acquired by bio - rad , inc .
each 20 l pcr reaction ( see above ) was loaded into an 8-channel , single - use consumable droplet generation cartridge .
60 l of oil containing emulsion - stabilizing , biocompatible surfactant was loaded into adjacent oil wells , and the microfluidic chip was loaded into a beta - series prototype droplet generator ( dg ) .
the dg applies a vacuum to the outlet well creating a pressure difference across the cartridge that simultaneously partitions the sample present in each of the 8 wells into 20,000 monodisperse droplets of accurately known volume .
the resulting water - in - oil emulsions were pipette - transferred from the outlet well to a 96-well polypropylene plate ( eppendorf ) , sealed with foil and then amplified to endpoint using a tetrad2 peltier thermal cycler ( bio - rad ) and the cycling protocol : 95 c for 10 min then 40 cycles of 95 c for 15 s and 60 c for 1 min ( 2.5 c / s ramp rate ) with a final 10 min hold at 98 c .
plates containing amplified droplets were loaded into an early - access , beta - version of the commercially available qx100 droplet reader ( bio - rad ) , which aspirates droplets from the 96-well plate , one well at a time , and streams them single - file ( 1,500 droplets / s ) past a two - color fam / vic ( fam and vic fluorescence dyes , life technologies corp . ) detector sampling at 100 khz .
discrimination between droplets that did not contain target ( negatives ) and those that did ( positives ) was achieved by applying a global fluorescence amplitude threshold .
for all of the mirna assays , the global fluorescence threshold was set at 4,000 relative fluorescence units ( rfus ) regardless of the assay efficiency
. concentration estimates (  ) were based on the fraction of droplets where amplification has occurred ( p ) modeled as a poisson distribution ( equation ( 1 ) ) .
because each droplet is an independent pcr reaction vessel of equal and defined volume , the droplets of technical replicates ( triplicate wells ) can be pooled to create a 
metawell. to compute total  95% confidence intervals about concentration estimates of metawells ( comprised of previously described poisson  95% cis and  real world error ; concentration differences between wells ) we applied the following meta - analysis techniques . given k replicates with concentrations m1 , m2 ,  mk and poisson variances v1 , v2 ,  vk , respectively , we define the weight ( w ) of replicate i as reciprocal of its variance as let m be the weighted average of concentrations and consider the following random variable that measures fluctuation of concentrations around this weighted mean : this is the sum of squares of approximately standard normal random variables and can therefore be approximated as a chi - squared distribution .
the mean of the distribution is the number of degrees of freedom ( df = k  1 ) .
if t is less than df , we say that there is no additional real - world variance .
if t is more than df , then it suggests there is additional real - world variance r = t  df .
as t is based on standard normal variables , we scale back r to r in original units after applying an appropriate correction factor : we can add r to poisson variance to give total variance for each replicate .
we redefine the weight of each replicate as : from which we can compute the total uncertainty around the metawell as the final estimate m is recomputed as weighted average of concentrations with new weights . by setting r to 0
this meta - analysis is a statistically rigorous method to test the existence of real - world error in a group of replicate droplet digital pcr wells ( us patent application 20130017551 ) .
all of these data - analysis methods are implemented in the quantasoft ( 1.1.1.0 ) data analysis package , installed with the droplet reader .
human serum samples from individuals with metastatic prostate cancer and prostate - cancer  negative controls were collected after written informed consent was obtained .
all participants signed a university of washington human subjects committee approved informed consent form for a peripheral blood draw and the research was approved and supervised by the university of washington and fred hutchinson cancer research center institutional review boards .
prostate - cancer  negative donors were recruited among individuals undergoing screening for prostate cancer and found to be negative by digital - rectal examination ( dre ) and serum psa analysis .
individuals with metastatic prostate cancer were recruited among previously diagnosed patients undergoing treatment at the university of washington ( seattle ) .
a smaller cohort of clinical specimens corresponding to n = 20 advanced prostate cancer cases and n = 20 healthy controls was analyzed ( previously , n = 25 each group ) .
the patient specimens studied here were distinct from ( not a subset of ) those we studied previously . using the population - variance data from the previous study , we performed a power calculation and determined that n = 12 individuals per group would result in 81% power to detect a difference in means of fourfold ( alpha = 0.05 , two tails ) .
we chose to analyze specimens from n = 20 individuals per group , as this was above the minimum predicted by the power calculation , feasible and simplified the technical work relative to the previous study .
the experimenter was kept blinded to case status ( i.e. , cancer versus control ) . for comparing the performance of ddpcr to real - time pcr in the clinical specimens
synthetic mir-141 standard curves used in this portion of the study were made using ms2 carrier rna , which leads to improved circulating mirna precision in real - time pcr analyses and therefore was included to provide the best possible real - time pcr performance to compare with ddpcr .
ddpcr showed superior precision ( supplementary fig . 8) , consistent with our results displayed in figure 1a , b and comparable limits of quantification ( supplementary table 7 ) for mir-141 in the analyses of these standard curves .
owing to limited serum rna volume for the large number of replicates needed for this study , we diluted the initial serum rna specimens by a factor of 1.5 . to allow a direct comparison of performance between ddpcr ( which uses bio - rad ddpcr supermix for probes , 186 - 3010 ) and standard circulating mirna real - time pcr ( which uses abi taqman universal pcr mastermix no ung , 4326614 ) , differences in the reagent volumes used between the bio - rad ddpcr and the standard circulating mirna real - time pcr protocols needed to be accommodated to achieve the same concentration of cdna in each comparable reaction across the methods ( supplementary fig . 7 ) .
as a result of these necessary adjustments , the concentration of input cdna analyzed per reaction for both ddpcr and real - time pcr methods was ultimately about tenfold lower than that used previously .
after reverse transcription , the cdnas corresponding to the sets of standard curves ( supplementary fig . 8) and the 40 clinical specimens ( fig .
2 ) were each split into six aliquots for subsequent pcr reactions ( see below ) .
three of these six cdna aliquots were added to abi master mix for subsequent real - time pcr , and the remaining three were added to bio - rad mastermix to conduct ddpcr and real - time pcr ( fig .
l ddpcr master mix for each sample was divided into three 20 l aliquots for analysis by ddpcr and three 5 l aliquots for analysis by real - time pcr .
the prepared 20 l standard real - time pcr master mix for each sample was divided into 3  5 l aliquots for analysis by real - time pcr ( fig . 2 and supplementary fig . 7 ) .
for the analysis of clinical specimens , ddpcr triplicates were combined into a single metawell before poisson analysis and recovery correction by caenorhabditis elegans recovery - yield mirna spike - ins . for real - time pcr , the concentration of analyte was calculated by ct comparison to the standard curve , and results were presented as the mean of the pcr triplicates corrected by c. elegans spike - ins .
statistical significance was determined by mann - whitney analysis ( 95% confidence intervals , two tails ) and receiver operating characteristic ( roc ) analysis was performed using prism 5.0c software .
supplementary figure 1| detailed schematic diagram of workflow to determine ddpcr operating characteristics relative to real time pcr .
standard curves were prepared by two -fold serial dilution ( supplementary table 1 ) of synthetic mirna stock oligonucleotides into either water or plasma rna solution background matrices .
each dilution series contained a no -template control ( matrix only ) as a final point .
reverse transcription ( rt ) of each standard curve was performed in triplicate followed by triplicate analysis of each rt by both ddpcr and real time pcr .
the gradient of shading in the plate row icons represents the standard curve concentration gradient .
the same homogenous reaction mixture ( cdna + mastermix + primers / probe ) was used for ddpcr and real time pcr , thus affording an analysis - platform - specific comparison of the two methods . for each of the six mirnas studied here , the entire workflow including preparation of standard curves
the graphs show side - by - side results generated from ddpcr and real time pcr analysis of dilution series of representative synthetic mirna oligonucleotides in water or plasma rna matrices . for each mirna ,
the results are organized by color , where each color represents one replicate ( preparation ) of the complete workflow outlined in supplementary figure 1 and shape represents pcr reactions de rived from individual rts ( rt1 = circle , rt2 = square , rt3 = triangle ) .
this amounts to nine pcrs per color for each point of the standard curve ( 1 preparation  3 rts  3 pcrs ) .
ddpcr data is presented as absolute concentration measured ( left -axis ) and relative concentration normalized to the mean average of all data for dilution point 1 ( right -axis ) .
as absolute quantification is not possible by real time pcr , results are displayed as relative concentration normalized to the mean average of all data for dilution point 1 and transformed from cycle -threshold ( ct ) space to linear space by pfaffl -analysis utilizing the empirically determined pcr efficiency of each assay .
relative ( normalized ) r esults between ddpcr and real time pcr analysis are therefore directly comparable on the graphs .
the statistical parameters used to generate the box and whisker plots ( gray ) for the combined data were median ( center line ) , 25   and 75 percentiles ( box ) , 10 and 90 percentiles ( whiskers ) . some clear outliers corresponding to failed replicate wells of real time pcr ( e.g. mirna-135b plasma rna matrix ) were excluded from subsequent computation of analytical figur es of merit given in supplemental figures 3 - 6 and supplemental tables 2 - 5 .
* note : the obscured dilution response for mir -16 is because this mirna is present endogenously in plasma rna at levels above the maximum synthetic oligonucleotide input .
coefficients of variation determined for both methods for each of the eleven oligonucleotide standard curve dilution points are displayed .
coefficient of variation measured at the level of pcr ( across -pcr % cv ) was determined by calculating the s.d . within each set of pcr triplicates ( n = 9 ) , dividing each of these by their respective mean values and then expressing the result as the overall mean of these individual % cv estimates .
% cv measured at the level of reverse transcription ( across - rt % cv ) was determined by calculating the s.d . across all pcr data derived from a set of rt triplicates ( n = 3 ) , dividing this by the corresponding mean and then expressing the result as the mean of these individual % cv estimates .
% cv measured at the level of preparation ( across - preparation % cv ) was determined by calculating the s.d .
across all the pcr data derived from the three preparative replicates , dividing the result by the corresponding mean and expressing this value .
the data clusters consistently above the line of identity , showing consistently higher % cv in the case of real time pcr . note : % cv for real time pcr was calculated using linear - scale values ( relative quantification ) rather than ct values in order to be directly comparable to ddpcr % cv as ddpcr data is ou tput on a linear scale .
therefore real time pcr % cv values displayed here are higher than if calculated using ct values .
coefficient of variation at the level of pcr ( across -pcr % cv ) was determined by calculating the s.d .
within each set of pcr triplicates ( n = 9 ) , dividing each of these by their respective mean values and then expressing the result as the overall mean and s.d . of these individual %
% cv at the level of reverse transcription ( across - rt % cv ) was determined by calculating the s.d . across all pcr data derived from a set of rt triplicates ( n = 3 ) , dividing this by the corresponding mean and then expressing the result as the mean and s.d . of these individual %
% cv at the level of prepar ation ( across - preparation % cv ) was determined by calculating the s.d .
across all the pcr data derived from the three preparative replicates , dividing the result by the corresponding mean and expressing this value .
note : % cv for real time pcr was calculated using linear - scale values ( relative quantification ) rather than ct values in order to be directly comparable to ddpcr % cv as ddpcr data is output on a linear scale .
therefore real time pcr % cv values displayed here are higher than if calculated using ct v alues .
supplementary figure 5| ddpcr shows consistently lower variation relative to real time pcr measured across - pcr , across - rt or across - preparation replicates ( water matrix ) .
mean % cv data from supplementary figure 2 presented in trendline format with ddpcr and real time pcr data plotted on opposing y -axes and a common x - axis .
note : % cv for real time pcr was calculated using linear - scale values ( relative quantification ) rather than ct values in order to be di rectly comparable to ddpcr % cv as ddpcr data is output on a linear scale . therefore real time pcr % cv values displayed here are higher than if calculated using ct values .
supplementary figure 6| ddpcr shows consistently lower variation relative to real time pcr measured across - pcr , across - rt or across - preparation replicates ( plasma rna matrix ) .
mean % cv data from supplementary figure 2 presented in trendline format with ddpcr and real time pcr data plotted on opposing y - axes and a common x - axis .
note : % cv for real time pcr was calculated using linear - scale values ( relative quantification ) rather than ct values in order to be di rectly comparable to ddpcr % cv as ddpcr data is output on a linear scale .
therefore real time pcr % cv values displayed here are higher than if calculated using ct values . supplementary figure 7| schematic illustrating the quantitative details of the workflow used in figure 2 .
rna was reverse transcribed as for analysis by ddpcr or a standard real time pcr method .
individual cdna aliquots were prepared for analysis using ddpcr master mix or standard real time pcr master mix on each of three separate days .
standard real time pcr master mix was analyzed only by real time pcr ( these reagents are not compatible with ddpcr analysis ) .
to enable appropriate quantitative comparison , the concentration of input rna ( as cdna ) for ddpcr and real time pcr was kept constant ( i.e. cdna concentration input into the pcr corresponded to 0.01 l rna solution per l pcr reaction ) .
supplementary figure 8| operating characteristics of ddpcr and real time pcr for measuring the circulating cancer biomarker mirna , mir-141 .
synthetic mirna oligonucleotide standard curves were prepared , reverse -transcribed and analyzed as described in methods and supplementary figures 2 and 7 .
results are presented as the concentration determined by poisson -metawell analysis of ddpcr triplicates or the mean concentration of three real time pcr replicates , respectively .
supplementary table 1| dilution protocol used to produce the standard curves for mirnas ( figure 1 , supplemental figures 2 and 8) and their predicted concentration in a pcr reaction assuming 100% rt efficiency and an accurate known concentration of the synthetic mirna supplied by the vendor .
supplementary table 2| coefficients of variation ( % cv ) for ddpcr and real time pcr ( water matrix ) .
coefficient of variation measured at the level of pcr ( across -pcr % cv ) was determined by calculating the s.d . within each set of pcr triplicates
( n = 9 ) , dividing each of these by their respective mean values and then expressing the result as the overall mean of the individual % cv estimates .
% cv at the level of reverse transcription ( across - rt % cv ) was determined by calculating the s.d . across all pcr data derived from a set of rt triplicates ( n = 3 ) , dividing this by the corresponding mean and then expressing the result as the mean of these individual % cv estimates .
% cv measured at the level of dilution series p reparation ( across - preparation % cv ) was determined by calculating the s.d . across all the pcr data derived from the three preparative replicates , dividing the result by the corresponding mean and expressing this value .
reduction in cv was calculated as the difference between the real time pcr cv ( cvrealtime ) and ddpcr cv ( cvddpcr ) divided by the real time pcr cv and expressed as a percentage : reduction in cv = ( cv real time  cvddpcr)/(cvreal time ) . note : % cv for real time pcr was calculated using linear - scale values ( relative quantification ) rather than ct values in order to be directly comparable to ddpcr % cv as ddpcr data is output on a linear scale .
therefore real time pcr % cv values displayed here are higher than if calculated using ct values .
supplementary table 3| coefficients of variation ( % cv ) for ddpcr and real time pcr ( plasma rna matrix ) .
coefficient of variation measured at the level of pcr ( across -pcr % cv ) was determined by calculating the s.d . within each set of pcr triplicates ( n = 9 ) , dividing each of these by their respective mean values and then expressing the result as the overall mean of the individual % cv estimates .
% cv at the level of reverse transcription ( across - rt % cv ) was determined by calculating the s.d . across all pcr data derived from a set of rt triplicates ( n = 3 ) , dividing this by the corresponding mean and then expressing the result as the mean of these individual % cv estimates .
% cv measured at the level of dilution series p reparation ( across - preparation % cv ) was determined by calculating the s.d .
across all the pcr data derived from the three preparative replicates , dividing the result by the corresponding mean and expressing this value .
reduction in cv was calculated as the difference between the real time pcr cv ( cvreal time ) and ddpcr cv ( cvddpcr ) divided by the real time pcr cv and expressed as a percentage : reduction in cv = ( cv real time  cvddpcr)/(cvreal time ) .
note : % cv for real time pcr was calculated using linear - scale values ( relative quantification ) rather than ct values in order to be directly comparable to ddpcr % cv as ddpcr data is output on a linear scale . therefore real time pcr % cv values displayed here are higher than if calculated using ct values .
operating characteristics based on ddpcr and real time pcr analysis of synthetic mirna oligonucleotides ( water matrix ) . limit of quantification ( loq ) was defined as the lowest concentration tested that remained above or equal to the limit of detection ( lod ) * and above or equal to the lower limit of linear range of the assay ( lllr)**. * lod was defined as = < x > bi + ksbi , were < x >
bi = mean of the no - template controls , sbi = standard deviation of no - template controls , k = 2.479 ( 99% confidence interval ) .
lod determinations here are likely to be biased in favor of real time pcr , as undetermined cts are set uniformly to 40 , artificially lowering sbi estimates for real time pcr . * * lllr was determined by runs - testing , removing successive lowest dilution points until the p - value was > 0.05 , indicating no significant deviation from linearity .
supplementary table 5| operating characteristics based on analysis of synthetic mirna oligonucleotides ( plasma rna matrix ) . limit of quantification ( loq ) was defined as the lowest concentration tested that remained above or equal to the limit of detection ( lod ) * and above or equal to the lower limit of linear range of the assay ( lllr)**. * lod was defined as = < x >
bi + ksbi , were < x > bi = mean of the no - template controls , sbi = standard deviation of no - template controls , k = 2.479 ( 99% confidence interval ) .
lod determinations here are likely to be biased in favor of real time pcr as undetermined cts = 40 , artificially lowering sbi estimates for real time pcr . * * lllr was determined by runs - testing , removing successive lowest dilution points until the p - value was > 0.05 , indicating no significant deviation from linearity .
* note : operating characteristics for mir-16 could not be determined in plasma rna matrix , as the endogenous abundance of mir -16 in plasma rna is higher than the maximum concentration of synthet ic oligonucleotide examined .
the mean average of the number of copies detected across all ddpcr data for a given mirna at the 250 -copy input dilution were divided by the predicted 250 input copies and expressed as a percentage .
supplementary table 7| operating characteristics of ddpcr and real time pcr for mir -141 measurement .
* lod determinations here are likely to be biased in favor of real time pcr as uniformly setting undetermined cts = 40 would artificially lower sbi estimates for real time pcr as previously described .
supplementary table 8| day - to - day variability of mir-141 quantification from clinical samples ( ddpcr master mix analyzed by ddpcr and real time pcr ) .
% cv : coefficient of variation calculated from replicates over 3 days ( percentage ) .
fold - change of % cv was calculated as the ratio of % cv for real time pcr results to % cv for ddpcr results .
supplementary table 9| day - to - day variability of mir-141 quantification from clinical samples ( ddpcr vs. standard real time pcr ) .
% cv : coefficient of variation calculated from replicates over 3 days ( percentage ) .
fold -change of % cv was calculated as the ratio of % cv for real time pcr results to % cv for ddpcr results .
 nanoliter - sized droplet technology paired with digital pcr ( ddpcr ) holds promise for highly precise , absolute nucleic acid quantification . 
 our comparison of microrna quantification by ddpcr and real - time pcr revealed greater precision ( coefficients of variation decreased by 3786% ) and improved day - to - day reproducibility ( by a factor of seven ) of ddpcr but with comparable sensitivity . 
 when we applied ddpcr to serum microrna biomarker analysis , this translated to superior diagnostic performance for identifying individuals with cancer . 
Online Methods
General procedure for generation of miRNA standard curves
Procedure for reverse transcriptionddPCR and reverse transcriptionreal-time-PCR
Procedure for the quantitation of miR-141 in clinical specimens
Supplementary Material