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Publication Supplemental Data

American Journal of Transplantation. 2004 Sept; 4(9):1475-89

Kidney Transplant Rejection and Tissue Injury by Gene Profiling of Biopsies and Peripheral Blood Lymphocytes

Stuart M. Flechner (1), Sunil M. Kurian (2), Steven R. Head (3), Starlette M. Sharp (2), Thomas C. Whisenant (3), Jie Zhang (4), Jeffrey D. Chismar (3), Steve Horvath (5), Tony Mondala (3), Timothy Gilmartin (3), Daniel J. Cook (1), Steven A. Kay (4), John R. Walker (4) and Daniel R. Salomon (2)

(1) Section of Renal Transplantation, Transplant Center A110, Cleveland Clinic Foundation, Cleveland, OH, (2) Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, (3) DNA Array Core Facility, The Scripps Research Institute, La Jolla, CA, (4) The Genomics Institute of the Novartis Research Foundation, San Diego, CA, (5) Departments of Human Genetics and Biostatistics, David Geffen School of Medicine, University of California, LA, CA

A major challenge for kidney transplantation is balancing the need for immunosuppression to prevent rejection, while minimizing drug-induced toxicities. We used DNA microarrays (HG-U95Av2 GeneChips, Affymetrix) to determine gene expression profiles for kidney biopsies and peripheral blood lymphocytes (PBLs) in transplant patients including normal donor kidneys, well-functioning transplants without rejection, kidneys undergoing acute rejection, and transplants with renal dysfunction without rejection. We developed a data analysis schema based on expression signal determination, class comparison and prediction, hierarchical clustering, statistical power analysis and real-time quantitative PCR validation. We identified distinct gene expression signatures for both biopsies and PBLs that correlated significantly with each of the different classes of transplant patients. This is the most complete report to date using commercial arrays to identify unique expression signatures in transplant biopsies distinguishing acute rejection, acute dysfunction without rejection and well-functioning transplants with no rejection history. We demonstrate for the first time the successful application of high density DNA chip analysis of PBL as a diagnostic tool for transplantation. The significance of these results, if validated in a multicenter prospective trial, would be the establishment of a metric based on gene expression signatures for monitoring the immune status and immunosuppression of transplanted patients.

MIAME Checklist:

Experiment Design

  • Type of Experiment: Control (Normal) vs. Transplant (Well-functioning) vs. Acute Rejection (Diseased) vs. Non-Rejection (Diseased)
  • Experimental Factors: Same as above in both Kidney Biopsy and Peripheral Blood Lymphocyte (PBL)
  • Number of Hybridizations: 62 (Biopsy and PBL combined)
  • Type of Reference: N/A
  • Hybridization Design: See table below
  • Quality Control: see RPT files
  • URL: http://www.scripps.edu/researchservices/dna_array/

Samples Used, Extract Preparation and Labeling

  • Origin of Biological Sample: All tissues are human
  • Manipulation of Biological Sample: Biopsy and Blood Drawing Protocols included in paper
  • Hybridization Preparation: TSRI DNA Array Core Facility RNA Extraction Protocol
  • Labeling: Standard Affymetrix Protocol (Affymetrix, Inc., Santa Clara, CA)
  • External Controls (Spike-ins): Standard Affymetrix Spike-in Controls (Affymetrix, Inc., Santa Clara, CA)

Hybridization Procedures and Parameters

  • Hybridization, Blocking and Washing: Standard Affymetrix Protocol (Affymetrix, Inc., Santa Clara, CA)

Measurement Data and Specifications

  • Image Quantitation: Affymetrix scanner to generate CEL file, dCHIP (PM-only model) to generate signal intensities

Array Design

  • General Array Design: Affymetrix HGU-95Av2 GeneChip (Affymetrix, Inc., Santa Clara, CA)

 

Sample Information:

*Patient ID and CEL files link to information in the NCBI Gene Expression Omnibus

 

 

Treatment Group
Patient ID
CEL File
Age
Sex
Immuno-suppression
Histo-pathology
LD/CAD
Scr(mg/dL)
Days Post TX
Normal Healthy Donor Biopsies
C1
GSM26836C1BX.CEL
38
F
-
-
-
0.8
-
C2
42
M
-
-
-
0.9
-
C3
35
F
-
-
-
0.6
-
C4
39
F
-
-
-
0.9
-
C5
39
M
-
-
-
1.2
-
C6
44
M
-
-
-
0.8
-
C7
36
M
-
-
-
1.2
-
C8
34
F
-
-
-
0.8
-
C9
50
F
-
-
-
0.6
-
Normal Healthy Donor Peripheral Blood Lymphocytes
C1
39
M
-
-
-
-
-
C2
-
-
-
-
-
-
-
C3
-
-
-
-
-
-
-
C4
-
-
-
-
-
-
-
C5
-
-
-
-
-
-
-
C6
-
-
-
-
-
-
-
C7
-
-
-
-
-
-
-
C8
-
-
-
-
-
-
-
Acute Rejection Biopsies
AR1
42
M
CsA/MMF/P
BanffIIA
CAD
12
285
AR2
28
M
FK/MMF/P
BanffIIA
LD
5.9
1467
AR3
18
M
CsA/MMF/P
BanffIA
CAD
2.2
119
AR4
28
F
FK/MMF/P
BanffIIA
CAD
1.5
366
AR5
26
F
CsA/MMF/P
Borderline
CAD
2
278
AR6
55
M
SRL/MMF/P
Borderline
CAD
2.9
68
AR7
35
M
SRL/MMF/P
BanffIA
CAD
2
184
Acute Rejection Peripheral Blood Lymphocytes
AR2
28
M
FK/MMF/P
BanffIIA
LD
5.9
1467
AR3
18
M
CsA/MMF/P
BanffIA
CAD
2.2
119
AR4
28
F
FK/MMF/P
BanffIIA
CAD
1.5
366
AR5
26
F
CsA/MMF/P
Borderline
CAD
2
278
AR6
55
M
SRL/MMF/P
Borderline
CAD
2.9
68
AR7
35
M
SRL/MMF/P
BanffIA
CAD
2
184
Well Functioning Transplant Biopsies
51
M
CsA/MMF/P
Normal
CAD
1.5
932
56
M
CsA/MMF/P
Normal
LD
1.3
911
52
M
CsA/MMF/P
Normal
CAD
1.2
902
31
F
CsA/MMF/P
Normal
LD
1.1
651
53
F
CsA/MMF/P
Normal
LD
1.1
689
32
M
CsA/MMF/P
Normal
LD
1.6
776
46
F
CsA/MMF/P
Normal
CAD
1.2
713
61
M
CsA/MMF/P
Normal
CAD
0.9
733
44
M
CsA/MMF/P
Normal
LD
1.8
718
21
M
CsA/MMF/P
Normal
CAD
1.5
674
Well Functioning Transplant Peripheral Blood Lymphocytes
38
M
CsA/MMF/P
-
CAD
1.4
461
57
F
FK/MMF/P
-
LD
1.3
42
65
M
CsA/MMF/P
-
CAD
1.5
213
65
F
FK/MMF/P
-
CAD
0.8
246
36
F
CsA/MMF/P
-
CAD
1.1
1278
68
M
CsA/MMF/P
-
CAD
1.7
376
39
M
SRL/MMF/P
-
CAD
0.9
36
61
F
CsA/MMF/P
-
CAD
0.9
1491
46
M
SRL/MMF/P
-
LD
1.2
81
Acute Graft Dysfunction without Rejection Biopsies
55
M
CsA/MMF/P
CNI toxicity
LD
1.7
456
38
M
FK/MMF/P
CNI toxicity
LD
2.3
155
61
M
SRL/MMF/P
ATN
LD
5.2
11
35
M
CsA/MMF/P
TN
CAD
6.3
16
22
F
FK/MMF/P
FSGS
LD
3.3
78
Acute Graft Dysfunction without Rejection Peripheral Blood Lymphocytes
55
M
CsA/MMF/P
CNI toxicity
LD
1.7
456
38
M
FK/MMF/P
CNI toxicity
LD
2.3
155
61
M
SRL/MMF/P
ATN
LD
5.2
11
43
M
CsA/MMF/P
CNI toxicity
CAD
3.8
262
35
M
CsA/MMF/P
TN
CAD
6.3
16
44
M
SRL/MMF/P
ATN
CAD
6.3
40
22
F
FK/MMF/P
FSGS
LD
3.3
78
-
-
-
-
-
-
-
CsA - Cyclosporine; MMF - Mycophenolate Mofetil; P - Prednisone; FK - Tacrolimus; SRL - Sirolumus; CAD - Cadaveric; LD - Live Donor; Scr - Serum Creatinine; ATN - Acute Tubular Nephropathy; TN - Tubular Nephropathy; CNI - Calcineurin Inhibitor; FSGS - Focal Segmental Glomerulosclerosis

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Tables and Figures:

Class Comparison Results:

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