File S1 - A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

Supporting information. Table S1, (A) Composition of Internal Standards Mixture (ISM) A–F. (B) Steps to calculate MYC/106 ACTB value for surgically removed, malignant sample 1 (SM1). To quantify the copy number for each target gene native template (NT) in a cDNA sample, 1) the ΔCq: [NT Cq - IS Cq]Sample for unknown sample and the average of two concentrations of ESM ΔCq: [NT Cq - IS Cq]ESM were calculated, 2) The corrected delta Cq was calculated as: [NT Cq - IS Cq]Sample - [NT Cq - IS Cq]ESM, 3) 2(− corrected delta Cq) was multiplied times the known number of input IS copies in the reaction to obtain the gene NT copy number, and 4) each target gene NT value was normalized to the ACTB loading control gene NT value, and presented as target gene NT molecules/106 ACTB molecules. ISM D (−13/−15) contains ACTB IS 10−13M/each target gene IS 10−15M that corresponds to ACTB IS 60000/each target gene IS 600 molecules. Figure S1, Schematic plot of experiment set up for 96 well plate. After dilution of pre-amplified PCR product containing cDNA and internal standards mixture (ISM), an aliquot of each diluted products was distributed into individual wells for 2nd round amplification for each individual gene native template (NT) and respective internal standard (IS) using gene-specific primers and probes. ISM C(−13/−15) was presented in the figure as an example, containing ACTB IS 10−13M/each target gene IS 10−15M corresponding to ACTB IS 60000 molecules/each target gene IS 600 molecules. The PCR amplification plots for ACTB from the two external standard mixtures (ESM), NT and IS each at 10−13M or NT and IS each at 10−14 M, are presented in one plot in the middle. Green is NT and red is IS in the plot. SM: surgically removed, malignant sample. NTC: no template control. Figure S2, Observed compared to expected positive PCR with limiting dilution PCR for each gene. Pre-amplification method was used for testing 9 replicates. Each of 10 dilution points of internal standards mixture (ISM) (40, 20, 10, 7, 4, 2, 1, 0.7, 0.4, 0.1 molecules) was multiplex pre-amplified nine times then observed PCR positivity for each gene at 2nd amplification. Figure S3, Observed compared to expected native template (NT) molecule values measured by two-color fluorometric assay in external standards mixture (ESM) dilution series samples. ESM (1/1 mixture of NT and internal standard (IS)) was serial 10-fold diluted from NT 10−11M/IS 10−11M to NT 10−17M/IS 10−17M and each dilution sample analyzed in triplicate. Figure S4, Observed compared to expected native template (NT) molecule values measured by two-color fluorometric assay in serially diluted synthetic NT relative to constant synthetic internal standard (IS) dilution series samples. ACTB, MYC, E2F1, or CDKN1A synthetic NT was serially diluted relative to constant synthetic IS, starting with 1/1 NT/IS mixture at 10−12 M. A, C, E, G: Linearity from 1/1 to 1/10-fold NT dilution. B, D, F, H: Linearity from 1/1 to 1/80-fold NT dilution. We assessed 1/1, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/12, 1/14, 1/16, 1/18, 1/20, 1/24, 1/28, 1/32, 1/36, 1/40, 1/48, 1/56, 1/64, 1/72, 1/80-fold dilutions of NT relative to constant IS. Data were analyzed with triplicate measurement. Figure S5, Observed compared to expected native template (NT) molecule values measured by two-color fluorometric assay in serially diluted synthetic internal standard (IS) relative to constant synthetic NT dilution series samples. ACTB, MYC, E2F1, or CDKN1A synthetic IS was serially diluted relative to constant synthetic NT starting with 1/1 of NT/IS mixture at 10−13 M. A, C, E, G: Linearity from 1/1 to 1/10-fold IS dilution. B, D, F, H: Linearity from 1/1 to 1/80-fold IS dilution. We assessed 1/1, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/12, 1/14, 1/16, 1/18, 1/20, 1/24, 1/28, 1/32, 1/36, 1/40, 1/48, 1/56, 1/64, 1/72, 1/80-fold dilution of IS relative to constant NT (one replicate measurement). Auto Cq could not be generated for the 1/56, 1/64, 1/72, 1/80-fold IS dilutions of ACTB. Table S2, Effect of external standards mixture (ESM) dilution on precision in measurement of each lung cancer diagnostic test (LCDT) genes by two-color fluorometric real-time assay. A serially diluted 1∶1 ratio of native template (NT): internal standard (IS) from 10−11M through 10−17M was analyzed in triplicate. Table S3, Effect of native template (NT) dilution relative to internal standard (IS) on precision in measurement of the lung cancer diagnostic test (LCDT) genes by two-color fluorometric real-time assay. For each gene serial dilution of synthetic NT up to 1/80-fold relative to constant IS was measured at each dilution. At each NT dilution points the compiled data across the four genes (ACTB, MYC, E2F1, CDKN1A) in triplicate measurements were presented. Figure S6, Effect of PCR reaction conditions on lung cancer diagnostic test (LCDT) gene expression values measured in cDNA with or without pre-amplification. A: pre-amplification. B: no pre-amplification. Surgically removed, formalin-fixed, paraffin-embedded, malignant sample 1(SM1) reverse transcribed with gene specific primers was used. The reference optimal PCR condition was 20 µl reaction volume (2V), 800 nM of primers (1Pm) and 200 nM of probes (1Pb). To test robustness, we reduced volume by half, and/or doubled primer or probe concentration in each of the two conditions (pre-amp or no pre-amp). Table S4, Histomorphological diagnosis of surgically removed, formalin-fixed, paraffin-embedded samples. Table S5, Total RNA sample quantity and purity assessment (A) (n = 20).
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