reverse transcription! RT 1! RT 2! RT 3!

Transcription

1 Supplementary Figure! Entire workflow repeated 3 times! mirna! stock! -fold dilution series! to yield - copies! per µl in PCR reaction! reverse transcription! RT! pre-pcr! mastermix!!!! 3!!! 3! ddpcr! Real time PCR! Synthetic mirnas analyzed:! mir-6! mir-35b! mir-! mir-5! mir-! mir-375! RT! pre-pcr! mastermix!!!! 3!!! 3! ddpcr! Real time PCR! RT 3! pre-pcr! mastermix! 3!!! 3!!! 3! ddpcr! Real time PCR! Nature Methods: doi:.3/nmeth.3

2 Supplementary Figure 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 ) 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 was repeated three times. Complete experimental details are given in Methods. Nature Methods: doi:.3/nmeth.3

3 Supplementary Figure! water matrix! plasma RNA matrix! ddpcr! Real time PCR! ddpcr! Real time PCR! mir-35b! mir-! mir-5! mir-! mir-375! Absolute copies/µl detected Absolute copies/µl detected Absolute copies/µl detected Absolute copies/µl detected Absolute copies/µl detected,.,.,.,., Absolute copies/µl detected Absolute copies/µl detected Absolute copies/µl detected Absolute copies/µl detected Absolute copies/µl detected,.,.,.,., mir-6! Absolute copies/µl detected, Absolute copies/µl detected, Nature Methods: doi:.3/nmeth.3 Absolute copies/µl detected Preparation RT Preparation RT Preparation RT 3 Preparation RT Preparation RT Preparation RT 3 Preparation 3 RT Preparation 3 RT Preparation 3 RT 3

4 Supplementary Figure mirna quantification by ddpcr and real time PCR. 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 and shape represents PCR reactions derived from individual RTs (RT = circle, RT = square, RT3 = triangle). This amounts to nine PCRs per color for each point of the standard curve ( 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 (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 and transformed from cycle-threshold (Ct) space to linear space by Pfaffl-analysis utilizing the empirically determined PCR efficiency of each assay. Real time PCR reactions of undetermined Ct were assigned Ct =. Relative (normalized) results 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), 5 th and 75 th percentiles (box), th and 9 th percentiles (whiskers). Some clear outliers corresponding to failed replicate wells of real time PCR (e.g. mirna-35b plasma RNA matrix) were excluded from subsequent computation of analytical figures of merit given in Supplemental Figures 3-6 and Supplemental Tables -5. *Note: The obscured dilution response for mir-6 is because this mirna is present endogenously in plasma RNA at levels above the maximum synthetic oligonucleotide input. Nature Methods: doi:.3/nmeth.3

5 NA Supplementary Figure 3! water! plasma RNA! across-preparations! Day to Day across-rts! to across-pcrs! to mir-! real-time PCR real-time PCR ddpcr ddpcr mir-375! real-time PCR real-time PCR ddpcr ddpcr mir-! real-time PCR real-time PCR ddpcr ddpcr mir-35b! real-time PCR real-time PCR ddpcr ddpcr mir-5! real-time PCR real-time PCR ddpcr ddpcr mir-6! real-time PCR real-time PCR ddpcr ddpcr Nature Methods: doi:.3/nmeth.3

6 Supplementary Figure 3 ddpcr displays consistently lower variation than real time PCR. 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 (acrosspreparation %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 output on a linear scale. Therefore real time PCR %CV values displayed here are higher than if calculated using Ct values. Nature Methods: doi:.3/nmeth.3

7 Supplementary Figure! water matrix! ddpcr! Real time PCR! plasma RNA matrix! ddpcr! Real time PCR! mir-! mir-375! mir-! mir-35b! mir-5! mir-6! across-pcrs! to across-rts! to Day across-preparations! to Day Nature Methods: doi:.3/nmeth.3

8 Supplementary Figure ddpcr and real time PCR variation is dependent upon analyte concentration. 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 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 and s.d. of these individual %CV estimates. %CV 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. 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. Nature Methods: doi:.3/nmeth.3

9 Supplementary Figure 5! across-preparations! Day to Day across-rts! to across-pcrs! to 3 mir-! mir-375! mir-! ddpcr! Real time PCR! mir-35b! 3 mir-5! mir-6! ddpcr! 6... Real time PCR! Nature Methods: doi:.3/nmeth.3

10 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 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 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. Nature Methods: doi:.3/nmeth.3

11 Supplementary Figure 6! across-preparations! Day to Day across-rts! to across-pcrs! to ddpcr! 3 mir-! 5 3 mir-375! 3 mir-! Real time PCR! mir-35b! 5 mir-5! 5 mir-6! ddpcr! 5 3 Real time PCR! Nature Methods: doi:.3/nmeth.3

12 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 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 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. Nature Methods: doi:.3/nmeth.3

13 Supplementary Figure 7! ddpcr! Real time PCR!. µl RNA per µl PCR reaction!. µl RNA per µl PCR reaction! 3 x µl! ddpcr reactions! 3 x 5 µl! real time! PCR reactions! µl BioRad! ddpcr! reaction mixture! µl ABI! real time PCR! reaction mixture! 6 µl frozen cdna! -> use 5µL! µl frozen! diluted cdna! -> use µl! 3 x 6µL cdna! aliquots! to be frozen! 3 x µl cdna! aliquots! to be frozen! µl cdna! 75.3 µl dilute! cdna! Dilute with! 7.3µL H! µl RT! 5 µl RT! µl RNA!.67 µl RNA! Nature Methods: doi:.3/nmeth.3

14 Supplementary Figure 7 Schematic illustrating the quantitative details of the workflow used in Figure. RNA was reverse transcribed as for analysis by ddpcr or a standard real time PCR method. cdnas were aliquotted and stored at - C. Individual cdna aliquots were prepared for analysis using ddpcr master mix or standard real time PCR master mix on each of three separate days. Aliquots of the ddpcr master mix solution were also analyzed by real time PCR. 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.µl RNA solution per µl PCR reaction). Nature Methods: doi:.3/nmeth.3

15 Supplementary Figure! ddpcr master mix! standard master mix! ddpcr! Real time PCR! Real time PCR! Absolute copies/µl detected Day Day Day 3 Nature Methods: doi:.3/nmeth.3

16 Supplementary Figure Operating characteristics of ddpcr and real time PCR for measuring the circulating cancer biomarker mirna, mir-. Synthetic mirna oligonucleotide standard curves were prepared, reversetranscribed and analyzed as described in Methods and Supplementary Figures 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. Whiskers represent -9 th distribution percentiles. Nature Methods: doi:.3/nmeth.3

17 Supplementary Table Dilution # (Tube Label) Transferred mirna (μl) Diluent (μl) mirna (copies/μl) Copies/μL in RT assay (when μl of dilution is added to a μl RT assay) Copies/μL in ddpcr (when μl of synthesized cdna are added to μl ddpcr assay) Supplied 6. x 99 6.x x x x 5 (96-well plate starts) Nature Methods: doi:.3/nmeth.3

18 Supplementary Table Dilution Protocol used to produce the standard curves for mirnas (Figure, Supplemental Figures and ) and their predicted concentration in a PCR reaction assuming % RT efficiency and an accurate known concentration of the synthetic mirna supplied by the vendor. Nature Methods: doi:.3/nmeth.3

19 Supplementary Table mirna Concentration (copies per µl in PCR) Across preparative replicates Real ddpcr time CV PCR CV Reduction in Across RT replicates Real time PCR CV ddpcr CV Reduction in Across PCR replicates Real time PCR CV ddpcr CV mir Mean reduction in mir-35b Mean reduction in 6 mir Reduction in Nature Methods: doi:.3/nmeth.3

20 Mean reduction in mir Mean reduction in mir Nature Methods: doi:.3/nmeth.3

21 Mean reduction in mir Mean reduction in Overall mean reduction in Nature Methods: doi:.3/nmeth.3

22 Supplementary Table 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 preparation (acrosspreparation %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 (CV real time ) and ddpcr CV (CV ddpcr ) divided by the real time PCR CV and expressed as a percentage: Reduction in CV = (CV real time CV ddpcr )/(CV real 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. Nature Methods: doi:.3/nmeth.3

23 Supplementary Table 3 mirna Concentration (copies per µl in PCR) Across preparative replicates Real ddpcr time CV PCR CV Reduction in Across RT replicates Real time PCR CV ddpcr CV Reduction in Across PCR replicates Real time PCR CV ddpcr CV mir-35b Mean reduction in mir Reduction in Mean reduction in Nature Methods: doi:.3/nmeth.3

24 mir Mean reduction in mir Mean reduction in mir Mean reduction in Nature Methods: doi:.3/nmeth.3

25 Overall mean reduction in CV () Nature Methods: doi:.3/nmeth.3

26 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 preparation (acrosspreparation %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 (CV real time ) and ddpcr CV (CV ddpcr ) divided by the real time PCR CV and expressed as a percentage: Reduction in CV = (CV real time CV ddpcr )/(CV real 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. Nature Methods: doi:.3/nmeth.3

27 Supplementary Table Limit of Quantification (copies per µl PCR) Limit of Detection (copies per µl PCR)* Lower Limit of Linear Range (copies per µl PCR)** mirna ddpcr Real time PCR ddpcr Real time PCR ddpcr Runs test P value Real time PCR Runs test P value mir mir mir mir-35b mir mir Nature Methods: doi:.3/nmeth.3

28 Supplementary Table 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 + ks bi, were <x> bi = mean of the notemplate controls, s bi = standard deviation of no-template controls, k =.79 (99% confidence interval). LOD determinations here are likely to be biased in favor of real time PCR, as undetermined Cts are set uniformly to, artificially lowering s bi estimates for real time PCR. ** LLLR was determined by runs-testing, removing successive lowest dilution points until the P-value was >.5, indicating no significant deviation from linearity. Nature Methods: doi:.3/nmeth.3

29 Supplementary Table 5 Limit of Quantification (copies/µl PCR) Limit of Detection (copies/µl PCR)* Lower Limit of Linear Range (copies/µl PCR)** mirna ddpcr Real time PCR ddpcr Real time PCR ddpcr Runs test P value Real time PCR Runs test P value mir mir mir mir-35b mir mir-6 NA* NA* NA* NA* NA* NA* NA* NA* Nature Methods: doi:.3/nmeth.3

30 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 + ks bi, were <x> bi = mean of the no-template controls, s bi = standard deviation of no-template controls, k =.79 (99% confidence interval). LOD determinations here are likely to be biased in favor of real time PCR as undetermined Cts =, artificially lowering s bi estimates for real time PCR. **LLLR was determined by runs-testing, removing successive lowest dilution points until the P- value was >.5, indicating no significant deviation from linearity. *Note: operating characteristics for mir-6 could not be determined in plasma RNA matrix, as the endogenous abundance of mir-6 in plasma RNA is higher than the maximum concentration of synthetic oligonucleotide examined. Nature Methods: doi:.3/nmeth.3

31 Supplementary Table 6 mirna Mean mirna copies Detection efficiency (%) per µl mir-6 5 mir-35b 9 79 mir- 7 5 mir-5 9 mir- 7 mir Nature Methods: doi:.3/nmeth.3

32 Supplementary Table 6 Absolute ddpcr detection efficiency. The mean average of the number of copies detected across all ddpcr data for a given mirna at the -copy input dilution were divided by the predicted input copies and expressed as a percentage. Nature Methods: doi:.3/nmeth.3

33 Supplementary Table 7 Limit of Quantification (copies per µl PCR) Limit of Detection (copies per µl PCR)* Lower Limit of Linear Range (copies per µl PCR)** master-mix ddpcr standard ddpcr standard ddpcr standard analysis Real Real Real ddpcr Real time ddpcr Real time Real time time time ddpcr time PCR PCR PCR PCR PCR PCR mir-.5.5 Nature Methods: doi:.3/nmeth.3

34 Supplementary Table 7 Operating characteristics of ddpcr and real time PCR for mir- measurement. LOQ, LOD and LLLR were defined as for Supplemental Tables and 3. *LOD determinations here are likely to be biased in favor of real time PCR as uniformly setting undetermined Cts = would artificially lower s bi estimates for real time PCR as previously described. Nature Methods: doi:.3/nmeth.3

35 Supplementary Table ddpcr (copies per µl in PCR) ddpcr master mix real time PCR (copies per µl in PCR) Sample Day Day Day Day Day Day CV Fold ID Status Mean s.d. %CV Mean s.d. %CV Number 3 3 Difference A Control B Case C Control D Case E Control F Case G Control H Case I Control J Case K Control L Case M Control N Case O Control P Case Q Control R Case S Control T Case U Control V Case W Control X Case Y Control Z Case AA Control BB Case CC Control DD Case Nature Methods: doi:.3/nmeth.3

36 3 EE Control FF Case GG Control HH Case II Control JJ Case KK Control LL Case MM Control NN Case Mean CV Fold Difference Nature Methods: doi:.3/nmeth.3

37 Supplementary Table Day-to-day variability of mir- quantification from clinical samples (ddpcr master mix analyzed by ddpcr and real time PCR). Results are presented as copies of mir- per µl in PCR reaction. s.d.: standard deviation. %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. Nature Methods: doi:.3/nmeth.3

38 Supplementary Table 9 ddpcr (copies per µl in PCR) Standard real-time PCR (copies per µl in PCR) Sample Day Day Day Day Day Day CV Fold ID Status Mean SD %CV Mean SD %CV Number 3 3 Difference A Control B Case C Control D Case E Control F Case G Control H Case I Control J Case K Control L Case M Control N Case O Control P Case Q Control R Case S Control T Case U Control V Case W Control X Case Y Control Z Case AA Control BB Case CC Control DD Case Nature Methods: doi:.3/nmeth.3

39 3 EE Control FF Case GG Control HH Case II Control JJ Case KK Control LL Case MM Control NN Case Mean CV Fold Difference 7 Nature Methods: doi:.3/nmeth.3

40 Supplementary Table 9 Day-to-day variability of mir- quantification from clinical samples (ddpcr vs. standard real time PCR). Results are presented as copies of mir- per µl in PCR reaction. s.d.: standard deviation. %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. Nature Methods: doi:.3/nmeth.3