Supplementary Figure 1: Experimental design. DISCOVERY PHASE VALIDATION PHASE (N = 88) (N = 20) Healthy = 20. Healthy = 6. Endometriosis = 33

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Clinton Marsh
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1 DISCOVERY PHASE (N = 20) Healthy = 6 Endometriosis = 7 EAOC = 7 Quantitative PCR (mirnas = 1113) a Quantitative PCR Verification of Candidate mirnas (N = 24) VALIDATION PHASE (N = 88) Healthy = 20 Endometriosis = 33 EAOC = 14 Serous Ovarian Cancer = 21 Quantitative PCR (mirnas = 24) a Selection of Candidate mirnas (N = 24) 1) Wilcoxon test 2) Logistic Regression model 3) Hosmer Lemeshaw Goodness -of-fit Test Supplementary Figure 1: Experimental design.

2 A. B. mir-132 mir-362-5p mir-1974

c. mir-132 CT 1 2 3 1 2 3 1 2 3 Patient 1 Patient 2 Patient 3 1 Supplementary Figure 2. Reliability of RNA extraction and reproducibility of RTqPCR techniques.

Left panel, serial dilution of synthesized mir-210 spiked into patient plasma prior to RNA extraction could be detected linearly.

3 c. mir-132 CT Patient 1 Patient 2 Patient 3 1 Supplementary Figure 2. Reliability of RNA extraction and reproducibility of RTqPCR techniques. A) Reliability of our RNA extraction technique was tested by adding various concentrations of synthesized mir-210 RNA to plasma samples, followed by RNA extraction, RT, and qpcr. Left panel, serial dilution of synthesized mir-210 spiked into patient plasma prior to RNA extraction could be detected linearly. Since the naked synthesized mirna is prone to degradation by RNases in plasma, synthesized mir-210 was mixed with LipoFectamine before spiked in plasma, which protected synthesized mir-210 from degradation; Right panel, dissociation curve analysis demonstrates the specificity of our PCR amplification. B) Reproducibility of our techniques, including RNA extraction, RT, and qpcr, was confirmed by performing three independent runs of three mirnas in six plasma samples. Similar CT values with only small variations are seen across each of the three runs. These results indicate that mirnas can be efficiently extracted and amplified from plasma and RT-qPCR results of plasma mirnas can be reliably compared across multiple samples. C) Reproducibility of our RT-qPCR assay using three consecutive plasma samples collected one month apart from each of three preoperative EAOC patients. These results indicate that plasma mirnas can be reliably measured in different plasma samples from the same patient.

4 A. Sample 1 Sample 2 CT CT Sample 3 CT

5 B CT Experimental Case Samples group Healthy Controls control Supplementary Figure 3. Reliability of RT-qPCR assays. A) Expression of mir- 16, 21, and 195 in three plasma samples collected in heparinized tubes and in matched serum samples collected in tubes without anticoagulant from EAOC patients. B) No difference is detected for plasma mir-132 expression (CT) between healthy control samples purchased from Innovative Research Labs and plasma samples from endometriosis and EAOC patients collected at Magee-Womens Hospital lby RT-qPCR. Average CT from each group is shown. Error bar, standard deviation.

6 3.45 log_m ir Log (Average Ct values) Mean CT 28.4 Mean CT 28.3 Mean CT 28.4 Mean CT Healthy 0=Normal Endometriosis 1=Endo 2=Serous SOC 3=EAOC Supplementary Figure 4. mir-132 demonstrates consistent CT values (ranging from 27 to 29.5.) across all categories of plasma samples (healthy controls, n=20; endometriosis, n=33; EAOC, n=14; and SOC, n=21), and was used for normalization of RT-qPCR results in this study.

7 A. B. C. C.

8 D. E. F. Supplementary Figure 5. Unsupervised hierarchical clustering analysis of samples based on the 23-miRNA expression profiles. A) Clustering of all four groups (healthy controls, n=20; endometriosis, n=33; EAOC, n=14; and SOC, n=21). Pair-wise clustering of B) endometriosis and EAOC, C) endometriosis and SOC, D) EAOC and SOC, E) healthy controls and EAOC, and F) healthy controls and SOC.

9 A. Norm vs. Endo log 2 (FC) Endo Norm -5 hsa.mir.16 hsa.mir.191 hsa.mir.195 B. Marker Norm vs. EAOC EAOC Norm log 2 (FC) hsa.mir.16 hsa.mir.191 hsa.mir.21 Marker

10 C. Endo vs. EAOC 5 10 EAOC Endo -5 0 log 2 (FC) hsa.mir.1274a hsa.mir.21 hsa.mir.362.5p Marker D. Norm vs. Serous 5 10 Serous Norm log 2 (FC C) -5 0 hsa.mir.16 hsa.mir.191 hsa.mir.4284 Marker

11 E. Endo vs. Serous 5 10 Serous Endo -5 0 log 2 (FC C) hsa.mir.1915 hsa.mir.362.5p hsa.mir.628.3p Marker F. EAOC vs. Serous Serous EAOC -5 0 lo og 2 (FC) 5 10 hsa.mir.191 hsa.mir.1975 hsa.mir.21 Marker Supplementary Figure 6. Box plots of top three differentially expressed mirnas in pair-wise comparisons. A) Endometriosis to health controls; B) EAOS to health controls; C) EAOC to endometriosis; D) SOC to health controls; E) SOC to endometriosis; F) SOC to EAOC. y-axis, log 2 of folder changes of plasma mirna expression (log 2 (FC)). Norm, healthy controls; Serous, SOC; Endo, endometriosis.

12 40 Mouse 1 40 Mouse Plasma CT Mouse 2 Tissue Mouse Plasma CT Mouse Tissue CT 30 Plasma CT Tissue CT Supplementary Figure 7. Lack of correlation for mirna expression between matching thi tumor tissue and plasma samples from five tumor-bearing LSL- Kras G12D/+ /Pten loxp/loxp mice. Expression of 10 mouse mirnas (mmu-mir-15b, 16, 21, 132, 191, 195, 362-5p, 652, 744, and 1274a) were examined by RTqPCR.

Lentiviral Delivery of Combinatorial mirna Expression Constructs Provides Efficient Target Gene Repression.

Supplementary Figure 1 Lentiviral Delivery of Combinatorial mirna Expression Constructs Provides Efficient Target Gene Repression. a, Design for lentiviral combinatorial mirna expression and sensor constructs.