Quantitative RT-PCR

Quantitative RT-PCR

Levels of CEACAM1, FKBP5, ACTB, VAMP5, and PLK2 mRNA were measured using TaqMan 5'-nuclease Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Three different primer sets were used to measure CEACAM1 transcript levels for the alternately-spliced long (L) form, the short (S) forms missing the cytoplasmic tail, and a subset of both long and short forms, respectively. cDNA was synthesized using Oligo(dT)20 (Invitrogen, Carlsbad, CA) andSuperscript III RT (Invitrogen, Carlsbad, CA),in accordance with themanufacturer's instructions.PCR reactions were prepared using the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), and cDNA derived from 20 ng of total RNA. Universal Human Reference RNA (Stratagene) was used to establish a standard curve, which was highly reproducible between assays (Pearson correlation coefficient >0.998 over a four-log (base 10) range). Relative abundance of the target transcripts was calculated by comparison to a standard curve, and normalized to the expression level ofTATA box binding protein-Associated Factor, RNA polymerase I, B (TAF1B). ABI assaycatalog numbers are as follows:

CEACAM1: Applied Biosystems (ABI) cat. # Hs00266109_m1 (detects splice variants 1L, 3L and 4L); Hs00989784_m1( detects 1S, 3S, and 4S); Hs00236077_m1(detects 4L, 4S, and 4C1); FKBP5: ABI cat # Hs00188025_m1; ACTB: ABIcat# Hs99999903_m1; VAMP5: ABI cat# Hs00198370_m1; PLK2: ABI cat# Hs00198320_m1; TAF1B: ABI cat. # Hs00374547_m1

Supporting Information: Figure Legends

Supporting Figure 3. Clustering of KD samples based on intrinsic gene expression patterns. The 65 samples from 20 patients in Figure 1 were re-organized using the 3 gene clusters identified as having the lowest (strongest) intrinsic scores. Letters a, b, and c correspond to the three intrinsic clusters shown in Figure 1. Horizontal bars mark samples from the same subject clustered together. Pre-treatment acute KD samples are in yellow; early post-treatment sub-acute samples in light blue; late post-treatment convalescent samples in purple.

Supporting Figure 4. Reproducibility of whole blood genome-wide transcript abundance patterns. Seven RNA samples from different KD patients were amplified, labelled, and hybridized to cDNA microarrays on two separate occasions. The set of 601 array elements that were present in both the final dataset for cohort 1 (Figure 1) and cohort 2 (Figure 4) were used to center and organize each of the two sets of seven samples. The two datasets were then jointly organized using hierarchical clustering. Each original RNA sample is indicated by a unique color. In all cases, the replicates of each sample were paired, indicating that relative patterns of expression were maintained across the two experiments.