St. Jude Research/Supplemental Data/ALL2/section F

F. TaqMan REAL-TIME RT-PCR (RT-PCR)

To further establish the validity of gene expression determined by microarray analyses we performed two additional experiments to validate the microarray analysis. (1) We determined gene expression by real-time RT-PCR (reverse transcription polymerase chain reaction) in four randomly selected post-treatment patient samples for three genes (AP1S2, BAX, and RBBP8) and (2) we determined the fold-change in gene expression by both RT-PCR for three genes in five paired patient samples (in both pre- and post treatment sample).

(1) We used TaqMan Universal PCR Master Mix Kit and the 7900-sequence-detection system (Applied Biosystems, Foster City, California). We designed primers and probes (Table 6) with Primer Express 2.0 software (Applied Biosystems, Foster City, California) and used the housekeeping gene RNase P (Applied Biosystems, Foster City, California) for normalization. One µg of total RNA was treated with DNase I and reverse transcribed using Superscript II Rnase H- reverse transcriptase and oligo dT primers (Invitrogen, Carlsbad, California). Additionally, we included controls that contained either no template or no reverse transcriptase, as negative controls in each run. We used aliquots (0.5 µl) of RT reaction mixture (20 µl) for quantification of AP1S1, BAX, RBBP8 and RNase P gene expression.

Table 6: Primers and probes used for real-time (TaqMan) RT-PCR

(1)	sequence	start	length
*AP1S2* (NM_003916) Forward Primer Reverse Primer Probe	5'-AAGCAATTGAGCAGGCTGATC-3' 5'-TCAGTCCAATTTCTTCAAGAACACTAC-3' 5'-6FAM-ACTGCAGGAGGAAGCTGAAACCCCA-TAMRA-3'	395 469 417	21 27 25
*BAX* (NM_004f324) Forward Primer Reverse Primer Probe	5'-TGGAGCTGCAGAGGATGATTG-3' 5'-GAAGTTGCCGTCAGAAAACATG-3' 5'-6FAM-AGAGGTCTTTTTCCGAGTGGCAGCTG-TAMRA-3'	221 315 267	21 22 26
*RBBP8* (NM_002894) Forward Primer Reverse Primer Probe	5'-GAACCCCCATGTCCGATACA-3' 5'-GGATGAGTTGAAGACTTGGAAACTTT-3' 5'-6FAM-ACATACTAAATTGGAGCACTCTGTGTGTGCAAATG-TAMRA-3'	838 936 868	20 26 35

(2)	sequence	assay-on-demand ID
*ATM* Probe	5'-6FAM-CAACGCGCAGGACTTCTGCACGGAC-TAMRA-3'	Hs00175892_m1
*BAX* Probe	5'-6FAM-AAACTGGTGCTCAAGGCCCTGTGCA-TAMRA-3'	Hs00180269_m1
*FOS* Probe	5'-6FAM-TATCAACCAAAGGCCTTCTTGTATC-TAMRA-3'	Hs00170630_m1

We checked primer quality by conventional PCR for amplification of the correct size and sequence of all transcripts. The total volume of the PCR reaction was 50 µl, containing 0.5 µl of RT-product, 400 nM each of the forward and reverse primers, 250 nM of probe, and 1X master mix. We used following thermal cycling parameters: two minutes at 50°C (activation of UNG enzyme to remove the carry-over PCR products), ten minutes at 95°C to activate AmpliTaq Gold DNA polymerase, 15 seconds at 95°C to denature and one minute at 60°C for annealing and extension, for a total of 45 cycles.

To estimate the amount of each of the three mRNAs in the four patient samples, we used linear regression analysis based on a standard curve representing six serial dilutions of cDNA made from the CEM human leukemia cells (American Type Culture Collection, Rockville, Maryland). In the standard curve, we plotted fluorescent signal intensities against the number of PCR cycles on a semi-logarithmic scale. Using CEM cDNA as standard, we achieved a high degree of linearity. We analyzed all unknown samples in triplicates in parallel with a standardization series using CEM cDNA. Based on the CT value and the corresponding standard curve the relative quantity of the specific mRNA for each sample was calculated. We plotted normalized and log transformed real-time RT-PCR gene expression level (AP1S2, BAX, and RBBP8) to the log-transformed signal from the Affymetrix MAS 5.0 output. The correlation between real-time RT-PCR and Affymetrix GeneChip was statistically significant (overall P=0.017, spearman rank test), as shown in Fig. 8 below and R² of 0.973, 0.798, 0.971 for AP1S2, BAX, and RBBP8, respectively, thereby confirming expression levels determined by the gene expression array.

Fig. 8: Real-time (TaqMan) RT-PCR vs. Affymetrix GeneChip®

Real-time (TaqMan) RT-PCR results are plotted versus expression levels determined by Affymetrix GeneChip® results for AP1S2, BAX and RBBP8, in four patients.

(2) We utilized the Assay-on-demandTM (part number 4331182, assay ID: ATM, Hs00175892_m1; BAX, Hs00180269_m1; FOS, Hs00170630_m1; Applied Biosystems, Foster City, California) according to the manufactures protocol (Table 6). To determine the relative quantity of gene expression of ATM, BAX, FOS in five pre- and five corresponding post-treatment patient samples we used the standard curve method and beta-actin, 18S-ribosomal RNA and RNAse P served as internal controls. We computed fold-change in expression for each gene based on post- to pre-treatment ratio as determined by RT-PCR, and we compared these fold-change values to the fold-change as determined by microarray analysis. As depicted in Fig. 9 below, the fold-change values determined by the two independent methods were highly correlated (R²=0.937).

Fig. 9: Fold-change RT-PCR versus Affymetrix GeneChip®

RT-PCR results are plotted versus fold-change determined by Affymetrix GeneChip® results for ATM, BAX and FOS in five patients

Fig. 10: Leave-one-out cross-validation of treatment classification.

Leave-one-out cross-validation using support-vector-machine (SVM) as the classifier, determined that prediction error rates were the smallest using linear discriminant analysis with variance (LDA) compared to ANOVA as the gene selection method. We constructed SVMs using the top-ranked discriminating genes selected by LDA or ANOVA. Leave-one-out cross-validation shows that classification error rate decreased with increasing number of genes, reaching zero only for fold-change in gene expression.

Table 7: Genes discriminating each treatment from all others.

Listed are the genes that were present in all three pair wise distinction calculations for each treatment group (all genes, P<0.05). There were 37 genes selected in common in each of the three pair wise comparisons of HDMTX vs. MP, HDMTX vs. HDMTX+MP and HDMTX vs. LDMTX+MP. There were 21 probe sets in common in the three pair wise comparisons for HDMTX+MP, 29 probe sets for MP alone and 9 probe sets for LDMTX+MP. The number assigned within each treatment group (e.g., HDMTX01) represents the rank of each gene by its distinction value in discriminating each treatment from the other treatments. The median fold-change in the individual treatment group (FC TX group) and the median fold-change in the other treatments combined (FC other TX groups) are shown for each gene, with minus (-) indicating genes that exhibited a decrease in expression, whereas a positive number indicates those genes that exhibited an increase in expression after treatment.

rank¹ by TX group	probe set ID	accession number	gene name	median FC TX group	median FC other TX groups
HDMTX01	37781_at	AB023138	neurexin 2	-1.7	1.1
HDMTX02	35926_s_at	AF004230	leukocyte immunoglobulin-like receptor	2.4	-1.7
HDMTX03	34751_at	AI970189	KIAA0997 protein	-1.4	1.5
HDMTX04	2067_f_at	L22475	BCL2-associated X protein	2.5	1.1
HDMTX05	41471_at	W72424	S100 calcium-binding protein A9 (calgranulin B)	4.6	-1.9
HDMTX06	38994_at	AF037989	STAT induced STAT inhibitor-2	-1.6	1.0
HDMTX07	31793_at	AL036554	defensin, alpha 3, neutrophil-specific	5.4	-1.4
HDMTX08	41096_at	AI126134	S100 calcium-binding protein A8 (calgranulin A)	4.2	-1.3
HDMTX09	38363_at	W60864	TYRO protein tyrosine kinase binding protein	1.9	-1.1
HDMTX10	41598_at	AA890010	SEC22, vesicle trafficking protein	-1.7	-1.2
HDMTX11	32749_s_at	AL050396	filamin A, alpha (actin-binding protein-280)	2.1	1.4
HDMTX12	31506_s_at	L12691	defensin, alpha 3, neutrophil-specific	9.4	-1.4
HDMTX13	39286_at	D64109	transducer of ERBB2, 2	-1.4	2.0
HDMTX14	35621_at	L77213	phosphomevalonate kinase	1.6	1.0
HDMTX15	41126_at	AA978353	phosphoserine aminotransferase	1.7	1.0
HDMTX16	402_s_at	X69819	intercellular adhesion molecule 3	1.8	1.1
HDMTX17	40362_at	X61498	nuclear factor of kappa light polypeptide gene enhancer	2.4	1.1
HDMTX18	36789_f_at	AF025534	leukocyte immunoglobulin-like receptor	1.8	-1.7
HDMTX19	37984_s_at	M57763	ADP-ribosylation factor 6	-1.9	-1.1
HDMTX20	38973_at	AB028943	HIC1-related gene on chromosome 22	-1.2	1.6
HDMTX21	679_at	J04990	cathepsin G	1.7	-1.5
HDMTX22	32227_at	X17042	proteoglycan 1, secretory granule	1.8	-1.4
HDMTX23	38999_s_at	M86707	N-myristoyltransferase 1	-1.1	1.5
HDMTX24	34965_at	AF031824	cystatin F (leukocystatin)	1.5	-1.2
HDMTX25	40877_s_at	AF041080	D15F37 (pseudogene)	1.1	1.5
HDMTX26	39119_s_at	AA631972	natural killer cell transcript 4	2.1	-1.3
HDMTX27	1403_s_at	M21121	small inducible cytokine A5 (RANTES)	1.7	-1.2
HDMTX28	34702_f_at	M27826	endogenous retroviral protease	1.1	-2.7
HDMTX29	33371_s_at	U59877	RAB31, member RAS oncogene family	1.4	-1.6
HDMTX30	37105_at	M16117	cathepsin G	2.4	-1.1
HDMTX31	31510_s_at	Z48950	H3 histone, family 3B (H3.3B)	-1.5	-1.2
HDMTX32	33661_at	U66589	ribosomal protein L5	-1.8	1.0
HDMTX33	40450_at	L09561	polymerase (DNA directed), epsilon	-1.6	1.7
HDMTX34	41360_at	AA044787	CCR4-NOT transcription complex, subunit 8	-1.5	1.0
HDMTX35	1402_at	M16038	v-yes-1 Yamaguchi sarcoma viral related oncogene	1.7	-1.1
HDMTX36	2000_at	U26455	ataxia telangiectasia mutated	2.6	1.2
HDMTX37	40520_g_at	Y00638	protein tyrosine phosphatase, receptor type, C	1.7	1.1

HDMTX+MP01	782_at	U93867	polymerase (RNA) III (DNA directed)	1.7	-1.2
HDMTX+MP02	40478_at	AL021396	hypothetical protein	2.3	1.1
HDMTX+MP03	39489_g_at	W27720	protocadherin 9	1.4	-1.5
HDMTX+MP04	37662_at	AI701164	ubiquitin-conjugating enzyme E2G 1	-1.8	1.1
HDMTX+MP05	491_at	U46116	HSPTPRG28 Human receptor tyrosine phosphatase	1.3	-2.1
HDMTX+MP06	33870_at	AB029005	chromosome 5 open reading frame 7	-1.7	-1.1
HDMTX+MP07	32257_f_at	AF003001	telomeric repeat binding factor (NIMA-interacting) 1	2.9	-1.1
HDMTX+MP08	322_at	D88532	phosphoinositide-3-kinase, regulatory subunit,	2.0	-2.1
HDMTX+MP09	40383_at	AB023200	gene from NF2/meningioma region of 22q12	2.2	-2.2
HDMTX+MP10	41667_s_at	AJ006068	dTDP-D-glucose 4,6-dehydratase	-1.6	1.3
HDMTX+MP11	39307_s_at	X81637	clathrin light chain b	-2.1	1.0
HDMTX+MP12	36729_g_at	M76446	adrenergic, alpha-1D-, receptor	2.2	-1.1
HDMTX+MP13	1814_at	D50683	transforming growth factor, beta receptor II	1.1	1.6
HDMTX+MP14	37563_at	AB007871	KIAA0411 gene product	2.3	-1.1
HDMTX+MP15	1368_at	M27492	interleukin 1 receptor, type I	2.5	-1.4
HDMTX+MP16	579_at	M95724	centromere protein C 1	-1.7	1.1
HDMTX+MP17	36514_at	U66469	cell growth regulatory with ring finger domain	-3.5	-1.4
HDMTX+MP18	41826_at	W28287	KIAA1467 protein	<-2.3	<1.1
HDMTX+MP19	33353_at	W26466	cDNA /gb=W26466	1.6	-2.3
HDMTX+MP20	33427_s_at	AF106861	Attractin	2.6	1.0
HDMTX+MP21	33958_at	T06733	cDNA /clone=HFBDX74	1.8	-1.7
HDMTX+MP22	32443_at	U28687	zinc finger protein 157 (HZF22)	1.4	-1.7

LDMTX+MP01	37036_at	AB002299	KIAA0301 protein	1.9	1.1
LDMTX+MP02	41218_at	AB018272	KIAA0729 protein	<-1.5	<1.1
LDMTX+MP03	37391_at	X12451	cathepsin L	-2.2	1.0
LDMTX+MP04	2081_s_at	L07032	protein kinase C, theta	-2.7	-1.1
LDMTX+MP05	38859_at	AL080141	secretory pathway component Sec31B-1	2.7	1.5
LDMTX+MP06	795_s_at	X66358	cyclin-dependent kinase-like 1 (CDC2-related kinase)	-2.6	-1.1
LDMTX+MP07	1186_at	D49493	growth differentiation factor 10	-2.7	-1.1
LDMTX+MP08	40607_at	U97105	dihydropyrimidinase-like 2	1.4	1.0
LDMTX+MP09	40377_at	AB014582	KIAA0682 gene product	2.8	1.3

MP01	35432_at	AF074723	RNA polymerase II transcriptional regulation mediator	2.4	-1.0
MP02	37244_at	AA746355	ubiquitin carboxyl-terminal esterase L3	1.8	-1.1
MP03	40942_g_at	W27026	vesicle-associated membrane protein-associated protein	-2.4	1.0
MP04	38390_at	Z34975	low density lipoprotein receptor defect C	1.7	1.0
MP05	38414_at	U05340	CDC20 (cell division cycle 20)	-1.1	-1.9
MP06	33134_at	AB011083	adenylate cyclase 3	1.5	1.0
MP07	35116_at	X80821	KIAA0874 protein	-1.8	1.1
MP08	39927_at	U17032	Rho GTPase activating protein 5	1.5	-1.2
MP09	1944_f_at	AF001359	DNA mismatch repair protein (hMLH1) alternatively spliced	2.3	-1.2
MP10	39199_at	W28661	cDNA /gb=W28661	<-2.4	<-1.4
MP11	34886_at	L02320	Radixin	-1.6	1.1
MP12	36694_at	AF043472	potassium voltage-gated channel, delayed-rectifier	1.8	-1.2
MP13	36297_at	X55544	activating transcription factor 1	1.4	-1.2
MP14	35227_at	U72066	retinoblastoma-binding protein 8	-1.5	1.0
MP15	36225_s_at	W27611	splicing factor proline/glutamine rich	1.7	-1.4
MP16	37077_at	D13243	pyruvate kinase L	-2.7	-1.3
MP17	39637_at	U14528	solute carrier family 26 (sulfate transporter)	2.5	-1.3
MP18	37624_at	M29458	Human carbonic anhydrase III	-2.9	1.1
MP19	37967_at	AF000424	lymphocyte antigen 117	-1.0	1.5
MP20	35005_at	AF051941	nucleoside diphosphate kinase type 6	1.6	-1.3
MP21	1106_s_at	M12959	T cell receptor alpha locus	-1.3	1.5
MP22	39775_at	X54486	serine (or cysteine) proteinase inhibitor,	-3.2	1.0
MP23	37553_at	D50863	testis-specific kinase 1	2.0	1.3
MP24	41188_at	W28186	putative integral membrane transporter	-2.4	1.0
MP25	41244_f_at	X80910	protein phosphatase 1, catalytic subunit, beta isoform	-2.1	-1.2
MP26	39674_r_at	AB011792	extracellular matrix protein 2	-2.4	-1.2
MP27	34927_at	M28826	CD1B antigen, b polypeptide	1.5	-1.6
MP28	32904_at	M28393	perforin 1 (pore forming protein)	-1.9	1.2
MP29	33490_at	L27071	TXK tyrosine kinase	1.8	-1.2

¹rank ordered by distinction values