CIRCRESAHA/2004/098145/R1 - ONLINE 1 Expanded Materials and Methods Validation by Semi-quantitative Real-Time Reverse Transcription PCR Expression patterns of 13 genes (Online Table 2), selected with respect to the central role in the proposed working model of AF (Figure 3) were validated by real-time SYBR-Green PCR 1 as described previously. 2 mrnas from all 30 tissue samples (10xpmAF, 20xSR) were used for RT-PCR experiments. Briefly, contaminating genomic DNA was removed from the isolated RNA using the DNA-free-kit from Ambion (Austin, Texas, USA). Total RNA content was then quantified by spectrophotometry. For in vitro reverse transcription, 200ng total RNA was preincubated at 70 C for 10min with random hexamer primer. Then, 1µl RNase-inhibitor (1.5U/µl; RNAsin; from Promega, Heidelberg, Germany), 1µl dntp-mix (containing each desoxyribonucleotide in a concentration of 10mM), 2µl 0.1M dithiothreitol, 4µl 5x reaction buffer (250mM Tris-HCl ph=8.4, 375mM KCl, 15mM MgCl 2 ; from Invitrogen, Karlsruhe, Germany) and 1µl Superscript II RNase H - -reverse transcriptase were added to obtain a total volume of 20µl and subsequently incubated at 42 C for 60 minutes. Finally, the enzyme was inactivated at 70 C for 15 minutes. Gene-specific primers and probes were designed using Primer 3 software (Applied Biosystems, Foster City, CA, USA) in order to amplify fragments of 70-150 base pairs in length close to the 3 -end of the transcript (Online Table 2). Real-time PCR oligonucleotide hybridization primers were purchased from Thermo Hybaid (Ulm, Germany). Gel electrophoresis and the configuration of the dissociation curves were used to assess the specificity of the amplicon. Real-time PCR was performed in triplicate for each sample with 10µl aliquot of diluted cdna (1:3) in the Mx4000 Detection System using the Brilliant SYBR Green QPCR Master Mix (containing SureStart Taq, dntps with dutps, optimized buffer and passive reference dye) according to the recommendations of Stratagene (La Jolla, CA, USA). PCR-amplification of cdna started with a "hot start"-activation of SureStart Taq polymerase at 95 C for 10 minutes, followed by 40 cycles of 15s denaturation at 95 C,
CIRCRESAHA/2004/098145/R1 - ONLINE 2 annealing for 60s at 58 C, and 10s elongation at 72 C. All experimental results for the samples with a coefficient of variation >10% were retested. To evaluate differences in gene expression, a method of relative quantification was used. 3 Several routinely used housekeeping genes (e.g. GAPD) were tested but not utilized due to their deregulation in pmaf. Ranking according to the least expression changes in a total of 213 array datasets of 161 patients accounting for major regional topology (left ventricle, left atrium, right ventricle, right atrium) and disease etiologies (dilated, ischemic, hypertrophic cardiomyopathies, non-failing ventricular tissue, atrial tissue from patients with sinus rhythm and permanent atrial fibrillation) detected ribosomal genes, notably RPL41 and RPL23A to exhibit the smallest changes in gene expression across all arrays. Likewise, some non-ribosomal transcripts like ubiquitin C (UBC) and tumor protein, translationally controlled 1 (TPT1) did not show deregulation in our datasets in SR and pmaf (unpublished data, 2005). After validation with SYBR-Green RT-PCR, RPL41 was selected as internal control in our experiments.
CIRCRESAHA/2004/098145/R1 - ONLINE 3 References Online Supplementary Material 1. Rajeevan MS, Vernon SD, Taysavang N, Unger ER. Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR. J Mol Diagn. 2001;3:26-31 2. Kääb S, Barth AS, Margerie D, Dugas M, Gebauer M, Zwermann L, Merk S, Pfeufer A, Steinmeyer K, Bleich M, Kreuzer E, Steinbeck G, Näbauer M. Global gene expression in human myocardium-oligonucleotide microarray analysis of regional diversity and transcriptional regulation in heart failure. J Mol Med. 2004;82:308-316. 3. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST ) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nuc Acids Res. 2002;30.
CIRCRESAHA/2004/098145/R1 ONLINE 4 Online Table 1. Patient demographics (Online Data Supplement). Patient Identifier GEO accession # HG-U133A GEO accession # HG-U133B Tissue Sex Age EF [%] Diagnosis Treatment SR-1 GSM40990 GSM41095 RA male 55 45 AoR T SR-2 GSM40991 GSM41096 RA female 54 74 MR A SR-3 GSM40992 GSM41097 RA female 72 60 CABG A, B, T SR-4 GSM40993 GSM41098 RA female 61 55 CABG A, B, T SR-5 GSM40994 GSM41099 RA male 67 77 CABG, MR, AoR A, B, T SR-6 GSM40995 GSM41100 RA male 52 65 MR none SR-7 GSM40996 GSM41101 RA male 43 60 CABG A, B, St SR-8 GSM40997 GSM41102 RA female 65 61 AoS T SR-9 GSM40998 GSM41103 RA female 59 74 MR, hyperthyroidism carbimazole SR-10 GSM40999 GSM41104 RA male 59 73 MR, DM, CABG L, D, St SR-11 GSM41000 GSM41105 RA male 68 57 CABG B, L, St SR-12 GSM41001 GSM41106 RA female 69 62 AoS, DM B, St SR-13 GSM41002 GSM41107 RA male 36 65 AoR L, S SR-14 GSM41003 GSM41108 RA male 60 75 MR A, B, T SR-15 GSM41004 GSM41109 RA male 75 65 MR A, B, St SR-16 GSM41005 GSM41110 RA female 74 82 MR A SR-17 GSM41006 GSM41111 RA male 60 47 AoS, AR A, T SR-18 GSM41007 GSM41112 RA male 72 45 AoS T SR-19 GSM41008 GSM41113 RA female 76 65 AoS, CABG A, B, T, St SR-20 GSM41009 GSM41114 RA male 75 65 CABG B, St AF-1 GSM40980 GSM41085 RA female 17 n/a DCM, MR, Marfan D, L AF-2 GSM40981 GSM41086 RA male 75 48 MR, AoR A, T AF-3 GSM40982 GSM41087 RA male 65 59 MR, AoR, DM B, D, L AF-4 GSM40983 GSM41088 RA female 71 47 MR A, L AF-5 GSM40984 GSM41089 RA male 53 53 MS B, D, T AF-6 GSM40985 GSM41090 RA female 58 52 AoS AIIR, B AF-7 GSM40986 GSM41091 RA male 70 38 MR A, B, T AF-8 GSM40987 GSM41092 RA male 75 52 MR, DM, CAD A AF-9 GSM40988 GSM41093 RA male 67 30 MR, DCM A, C, L AF-10 GSM40989 GSM41094 RA male 78 73 MR AIIR, L LV-1 GSM41010 GSM41115 LV female 50 n/a non-failing donor none reported LV-2 GSM41011 GSM41116 LV female 53 n/a non-failing donor none reported LV-3 GSM41012 GSM41117 LV female 62 n/a non-failing donor none reported LV-4 GSM41013 GSM41118 LV female 38 n/a non-failing donor none reported LV-5 GSM41014 GSM41119 LV male 42 n/a non-failing donor none reported SR = sinus rhythm, AF = atrial fibrillation, LV = left ventricle, RA = right atrium, EF = left ventricular ejection fraction, as determined by left ventriculography; AoR = aortic regurgitation; AoS = aortic stenosis; MR = mitral regurgitation; MS = mitral stenosis, CABG = coronary artery bypass graft; CAD = coronary artery disease; DCM = dilated cardiomyopathy; DM = diabetes mellitus, n/a = not available. A = ACE-inhibitor; AIIR = Angiotensin II receptor antagonist, B = beta-blocker; C = calciumantagonist; D = digitalis; L = loop-diuretic; S = spironolactone; T = thiazide-diuretic; St = statin.
CIRCRESAHA/2004/098145/R1 - ONLINE 5 Online Table 2. Oligonucleotides used for RT-PCR (Online Data Supplement). Name Sequence of primer Annealing temperature Reference sequence CALR Forward: 5 GAA GAG ATG GAC GGA GAG TGG 3' 60 C BE251303 Reverse: 5' GGG TTG TCA ATT TCT GGG TG 3' CREB Forward: 5 CGA GAA CCA GCA GAG TGG AGA TGC 3' 63 C NM_004379 Reverse: 5' TAG AGT TAC GGT GGG AGC AGA TGA TG 3' DSCR1 Forward: 5 AAC TTC AGC AAC CCC TTC TCC GC 3' 63 C NM_004414 Reverse: 5' GGG TCG CAT CTT CCA CTT GTT TCC 3' FBN1 Forward: 5 ATG GGA TCT TAC CGC TGT CTG 3' 60 C NM_000138 Reverse: 5' TCA CAG GCT TTC CCG TCA G 3' GAPD Forward: 5 AGC CAC ATC GCT CAG ACA CC 3' 60 C NM_002046 Reverse: 5' GTA CTC AGC GCC AGC ATC G 3' GSK3A Forward: 5 CAG TGG CGA GAA GAA AGA CGA GC 3' 61 C L40027 Reverse: 5' GAT GTA GGC CAA GCT GCG GAA G 3' ITPR1 Forward: 5 CGG AGC AGG GTA TTG GAA C 3' 60 C U23850 Reverse: 5' CTT CAG GCA CAG AGA CCA GG 3' KCNK3 Forward: 5 GGC TTC TTC TCG TGC ATC AG 3' 60 C BF513479 Reverse: 5' AGG GTG ATG AAG CAG TAG TAG TAG G 3' MAPK1 Forward: 5 AAG ACA CAA CAC CTC AGC AAT G 3' 60 C NM_002745 Reverse: 5' GTT GAG CAG CAG GTT GGA AG 3' NFATc3 Forward: 5 TTC CAT CTT TGC CTG TGC C 3' Reverse: 5' GCA GAT GGG ATG AGG TCA CC 3' 60 C U85430 NFKB1 Forward: 5 CAG GAG AGG ATG AAG GAG TTG TGC C 3' 58 C M55643 Reverse: 5' GCC AGA GTA GCC CAG TTT TTG TCT G 3' PPP3CA Forward: 5 AGG AGG GAA GGC TGG AAG AG 3' 60 C AI827550 Reverse: 5' GAA GAG GTA GCG AGT GTT GGC 3' STAT5B Forward: 5 CAT GGC TGT GTG GAT ACA AGC TCA G 3' Reverse: 5' GAT CTA CTG AGT CCC ATG CTT GGC 3' 61 C NM_012448
CIRCRESAHA/2004/098145/R1 - ONLINE 6 Online Table 3. List of all 1.434 transcripts differentially expressed in SR and pmaf (Microsoft Excel -format; Online Data Supplement). Transcripts are ranked according to their statistical significance (represented by the q-value). In addition to Affymetrix probe set IDs and gene names, Unigene accession numbers are also given for each gene. By activating the Auto Filter function of Microsoft Excel, differentially expressed transcripts can be grouped into transcripts predominantly expressed in left ventricle (LV) and right atrium (RA). Online Table 4. Gene Ontology classes for differentially expressed transcripts of cellular components, biological processes, and molecular function (level 5; Microsoft Excel - format; Online Data Supplement). Only classes including more than 1% of transcripts of the respective GO level or showing statistically significant changes (p<0.05) are given. The respective p-values for the comparison between the different regions are also indicated (pvalues <0.05 are highlighted by yellow background). Online Table 5. Myocardial transcripts for cellular component, biological process and molecular function (GO, level 5). The list was generated by means of the FatiGO software tool by searching the Unigene accession numbers (Online Data Supplement).
CIRCRESAHA/2004/098145/R1 - ONLINE 7 Online Figure 1. Unsupervised clustering using euclidean distance and an average linkage algorithm for all 1.434 transcripts regulated in pmaf (Online Data Supplement). Rows represent individual transcripts, columns represent different samples. Transcripts with elevated or repressed expression are represented in shades of yellow and blue, respectively. Of note, atrial pmaf samples cluster with ventricular samples.