A System Approach

Variance Analysis For Multiplexed qPCR Samples: A System Approach

Designed by Geoffrey Dennis and Concept Proofed by Hugo DiGiulio

Variance Analysis of Multiplexed Systems: Individual vs. Whole

• Multiplexing 2 gene targets for quantitation in single well format

• Typical basic analysis of individual amplisets includes CV (RSD), CI, relevant t-test

• Comparison between systems can be done by ANCOVA, t-test, test, etc.

• By use of dilution series, the multiplex can be set up as a matrix and the system variance can be analyzed as a single unit

– This is done by calculating a %CV based on frequency of particles

– using %CV calculated by frequency of particles provides clarity on the decision to accept or not accept the outcome of a sample

Example of Variance Analysis of Individual Parts

Gene 1 Gene 2 Gene 3 DCt Gene 1 Gene 2 Gene 31000000 19.97 19.68 19.95 -3.91 -3.60 -3.71 Slope

300000 21.38 21.15 21.60 1.65 43.13 40.99 42.01 Intercept100000 23.50 22.71 23.44 1.83 0.253 0.22 0.14 Error30000 25.72 24.58 25.09 1.66 0.142 0.105 0.174 ANCOVA10000 27.69 26.76 27.14 2.043000 29.13 28.23 28.88 1.751000 31.17 30.22 30.87 1.98

300 33.43 31.85 32.80 1.93100 35.55 34.01 34.72 1.92

NTC Neg Neg Neg

Gene 1 Gene 2 Gene 3Sample dilution 1 24.03 23.28 23.83Sample dilution 2 27.52 26.94 27.40Sample dilution 1 3.10E+09 3.30E+09 3.18E+09Sample dilution 2 3.96E+09 3.17E+09 3.47E+09

17.4% 2.7% 6.1% %CV

Resulting CqResulting c/mL

Example of Variance Analysis of System as Whole

%CVWhole = 2 – 1 + 2)

Gene 1 Gene 2 Gene 3Sample dilution 1 24.03 23.28 23.83Sample dilution 2 27.52 26.94 27.40Sample dilution 1 3.10E+09 3.30E+09 3.18E+09Sample dilution 2 3.96E+09 3.17E+09 3.47E+09

17.4% 2.7% 6.1% %CV

Sample dilution 1 0.32 0.34 0.33Sample dilution 2 0.37 0.30 0.33

0.7 0.7 0.70.0 0.0 0.0

5.5% %CV

Resulting CqResulting c/mL

Frequency of ParticlesCalculated Lambdas

Example of Variance Analysis of System as Whole: Accept or Reject?

• While 36% is typically acceptable in c/mL for qPCR, calculating %CV by using frequency of particles in a matrix may result in a need to repeat the sample

• Observed acceptance tolerance was at a maximum of 10% CV compared to controls

Sample A Gene 1 Gene 2 Gene 3Sample dilution 1 3.10E+09 3.30E+09 3.18E+09Sample dilution 2 3.96E+09 3.17E+09 3.47E+09

17.4% 2.7% 6.1% %CV


0.7 0.7 0.70.0 0.0 0.0

5.5% %CV

Sample B Gene 1 Gene 2 Gene 3Sample dilution 1 1.88E+09 2.07E+09 3.00E+09Sample dilution 2 3.12E+09 3.48E+09 3.46E+09

35.0% 36.0% 10.1% %CV


0.6 0.7 0.70.0 -0.1 -0.1

12.7% %CV

Resulting c/mL


Resulting c/mL


Variance Analysis of a System as a Whole

• For each additional ampliset, – %CV = (2+N additional sets)- 1 + 2)

CV indicates direction of inconsistency in system (but remember, since 2+N used, the result indicates the inverse of the direction!)

Variance Analysis System Dynamics

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.6 0.62 0.64 0.66 0.68 0.7 0.72 0.74

Series1

Series2

Series3

Series4

Series 1 0.06Series 2 0.13Series 3 -0.02Series 4 -0.18

System CV

The arrows show how signal changes are reflected with this analysis

Rating Complex Systems

5`5`

Capped RNA miRNAFor 1 Probe

Rev

For 2

RT Primer

For Probe

Rev

Rating Complex Systems ContinuedRNA Cap Detection Set 1 Set 2Sample dilution 1 3.10E+09 3.30E+09Sample dilution 2 2.88E+09 2.07E+09Sample dilution 3 3.12E+09 3.48E+09Sample dilution 4 3.96E+09 3.17E+09

Sample dilution 1 0.48 0.52Sample dilution 2 0.58 0.42Sample dilution 3 0.47 0.53Sample dilution 4 0.56 0.44

1.00-0.10 1.001.00 0.11-0.08 9.9% %CV

Lambdas

Resulting c/mL

Frequency of Particles

Rating Complex Systems Continued

miRNA Evaluation miRNA 1 miRNA 2 miRNA 3 miRNA 4Sample dilution 1 3.10E+11 3.30E+11 3.18E+11 4.64E+11Sample dilution 2 3.96E+11 3.17E+11 3.47E+11 4.59E+11

17.4% 2.7% 6.1% 0.7%

Sample dilution 1 0.22 0.23 0.22 0.33Sample dilution 2 0.28 0.22 0.24 0.32

0.5 0.5 0.5 0.6 0.6 0.6

0.0 0.0 0.0 0.0 0.0 0.03-sum 's -2.2% %CV

Conclusion

• In conclusion, using %CV based on matrices calculated by frequency of particles provides clarity on the decision to accept or not accept the outcome of a sample

A System Approach

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