Genomic DNA was extracted from colorectal carcinoma tissue and adjacent non-tumor tissue in whole-tissue sections of FFPE tissue blocks using QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). Custom TaqMan primer/probe sets (Applied Biosystems, San Diego, CA) for the 16S ribosomal RNA gene DNA sequence of Fusobacterium nucleatum and for the reference gene, SLCO2A1 were used as previously described.24 (link) The primer/probe set for Fusobacterium nucleatum was designed to target the nusG gene of Fusobacterium nucleatum, and it has been demonstrated that the amount of Fusobacterium nucleatum measured by the quantitative PCR assay highly correlates with that measured by using transcriptome sequencing data (Pearson’s r = 0.97).24 (link) Each reaction contained 80 ng of genomic DNA and was assayed in 20 µL reactions containing 1× final concentration TaqMan Environmental Master Mix 2.0 (Applied Biosystems, San Diego, CA) and each TaqMan Gene Expression Assay (Applied Biosystems, San Diego, CA), in a 96-well optical PCR plate. Amplification and detection of DNA was performed with the StepOnePlus Real-Time PCR Systems (Applied Biosystems, San Diego, CA) using the following reaction conditions: 10 min at 95°C and 45 cycles of 15 sec at 95°C and 1 min at 60°C. The primer and probe sequences for each TaqMan Gene Expression Assay were as follows: Fusobacterium nucleatum forward primer, 5’-CAACCATTACTTTAACTCTACCATGTTCA-3’; Fusobacterium nucleatum reverse primer, 5’-GTTGACTTTACAGAAGGAGATTATGTAAAAATC-3’; Fusobacterium nucleatum FAM probe, 5’-GTTGACTTTACAGAAGGAGATTA-3’; SLCO2A1 forward primer, 5’-ATCCCCAAAGCACCTGGTTT-3’; SLCO2A1 reverse primer, 5’-AGAGGCCAAGATAGTCCTGGTAA-3’; SLCO2A1 VIC probe, 5’-CCATCCATGTCCTCATCTC-3’. In colorectal carcinoma cases with detectable Fusobacterium nucleatum, the cycle threshold (Ct) values in the quantitative PCR for Fusobacterium nucleatum and SLCO2A1 decreased linearly with the amount of input DNA (in a log scale) from the same specimen (r2 > 0.99; Figure 1A ). The inter-assay coefficient of variation of Ct values from the same specimen in five different batches was 1% or less for all targets in our validation study using seven colorectal carcinomas (eTable 1 in the Supplement ).
Each specimen was analyzed in duplicate for each target in a single batch, and we used the average of the two Ct values for each target. Spearman’s rank-correlation coefficients between the two Ct values (in duplicated runs) in each of cases with detectable target amplification in the quantitative PCR assays for Fusobacterium nucleatum (n = 76) and SLCO2A1 (n = 598) were 0.95 and 0.92, respectively. The amount of Fusobacterium nucleatum in each specimen was calculated as a relative unitless value normalized with SLCO2A1 using the 2−ΔCt method (where ΔCt = “the average Ct value of Fusobacterium nucleatum” - “the average Ct value of SLCO2A1”) as previously described.32 (link)
Each specimen was analyzed in duplicate for each target in a single batch, and we used the average of the two Ct values for each target. Spearman’s rank-correlation coefficients between the two Ct values (in duplicated runs) in each of cases with detectable target amplification in the quantitative PCR assays for Fusobacterium nucleatum (n = 76) and SLCO2A1 (n = 598) were 0.95 and 0.92, respectively. The amount of Fusobacterium nucleatum in each specimen was calculated as a relative unitless value normalized with SLCO2A1 using the 2−ΔCt method (where ΔCt = “the average Ct value of Fusobacterium nucleatum” - “the average Ct value of SLCO2A1”) as previously described.32 (link)
