Fathman Lab In the Division of Immunology & Rheumatology

RoadMap of NOD T1D

Under funding from the NIH (U19 DK61934), we have begun an analysis of the gene expression changes that are seen during the course of NOD disease by comparing mRNA obtained from multiple tissues from NOD mice during the time course of disease progression to organ matched mRNA obtained from NOD.B10 mice. This analysis should allow the identification of gene expression changes driven by inflammation as a result of the disease associated NOD MHC compared to the non-disease associated NOD.B10 MHC in otherwise isogenic mice. These studies are described below and the data obtained to date can be accessed as described below. As additional data are obtained from these studies, they will be made available on this web site.

Figure 1 A

Mice

NOD/LtJ (NOD) and NOD.B10Sn-H2b/J (NOD.B10) breeders were purchased from The Jackson Laboratory (Bar Harbor , ME) and were bred and maintained under pathogen-free conditions according to institutional guidelines under approved protocols in the Stanford Medical Center’s Department of Comparative Medicine (Stanford, CA). Only female mice were used for this study. 

Study Design

Six Groups of NOD female mice were sacrificed at 10 days (Group A), 4 weeks (Group B), 8 weeks (Group C), 12 weeks (Group D), 16 weeks (Group E), or 20 weeks of age (Group F). At each time point, the following four tissues were removed from respective mice and three were immediately processed for mRNA analysis of gene expression: pancreatic lymph nodes (PLN), spleen (SPL) and peripheral blood cells (PBC), the pancreata were frozen for future laser capture microscopic extraction of the islets. Non-fasting blood glucose levels were also measured with a One Touch Ultra glucometer (Johnson & Johnson, Milpitas, CA) when mice were sacrificed at 8 weeks, 12 weeks, 16 weeks or 20 weeks of age . Two Groups of 10 NOD.B10 female mice were sacrificed at 10 days and 20 weeks of age, and the same tissues were extracted. The two groups of NOD.B10 samples were merged (10 days + 20 weeks of age) for each tissue and used as control for gene expression levels from non-inflamed tissue. Gene (mRNA) expression in the PLN, SPL, PBC and pancreatic islets has been (or will be) analyzed using the 41K Whole Mouse Genome (60-mer) Oligo Microarray Kit (Agilent Technologies, Palo Alto, CA)

RNA Purification

Tissues were homogenized rapidly in 1ml of TRIzol Reagent (Invitrogen Corp., Carlsbad, CA ) and then frozen on dry ice in aliquots of 0.5 ml. For PBC, blood collected through periorbital bleeding was immediately added to 4 ml of Red Blood Cell Lysing Buffer (Sigma-Aldrich, St. Louis , MO ). RNA purification of individual samples of cells from the PLN, SPL and PBC followed the RNeasy Kit Protocol ( Qiagen , Valencia , CA). Quantitation of RNA samples was performed using NanoDrop spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington , DE ) and integrity of RNA samples was assessed by Agilent biosizing gel separation (Agilent Technologies).

cRNA Amplification & Fluorescent Labeling

cRNA amplification and fluorescence labeling was performed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). For the individual NOD mice, one target for each sample (PLN and SPL) was prepared. For the NOD.B10 control, pooled samples from PLN and SPL from all 20 mice (10 days + 20 weeks of age) were used as a tissue specific control. The cRNA synthesis involves a single amplification, for two color labeling to create ample cyanine 3 (for NOD) and cyanine 5 (for NOD.B10) labeled cRNA. In this technique a primer, which contains poly dT and a T7 polymerase promoter, is annealed to the poly A+ RNA (mRNA). Reverse transcriptase is added to the reaction to synthesize the first and second strands of cDNA (to create double stranded DNA). Fluorescence labeled anti-sense cRNA is next synthesized with T7 Polymerase, which simultaneously incorporates cyanine 3 or cyanine 5 labeled cytidine triphosphate (CTP). Both samples can be combined and hybridized on to the Agilent 41K whole mouse genome (60-mer) oligo microarray slide.

Hybridization & Feature Extraction

Using the Agilent Two-Color Microarray-Based Gene Expression Analysis protocol, the fragmentation mix consisting of 750ng of cyanine 3 labeled amplified cRNA , 750ng of cyanine 5 labeled amplified cRNA, 50μ of 10X blocking agent (Gene Expression Hybridization Kit; Agilent Technologies), 10μl of 25X fragmentation buffer (Agilent Technologies) and nuclease free water to a total volume of 250μl, was incubated for 30 minutes at 60 degree Celcius and then 250 μl of 2X hybridization buffer was added to stop the fragmentation reaction. The 500μl solution was then pipetted onto the microarray and incubated in oven rotator at 60 degree Celcius for 17 hours.

Due to a high level of ozone in the lab, the Agilent Stabilization and Drying Solution protocol was used. Washing conditions include 1st, 2nd, Acetonitrile for 1 minute each and the 3rd wash for 30 seconds on a magnetic stir plate. The arrays were then scanned using a Agilent Microarray Scanner laser based detection system. Background subtracted signal data (BGSub Signal: raw spot signal - background signal) and normalized expression ratios: Log 10 (NOD processed signal / NOD.B10 processed signal) were obtained from images using the Agilent Feature Extraction software version 8.5. These signal data were output, together with the Agilent probe name, GenBank identification number and gene description, to a text file.

Data Analysis

Normalized gene expression ratios: Log10 (NOD processed signal / NOD.B10 processed signal) (Log Ratio) were used for data analysis calculation.

Graphmaker for RoadMap Data

Download Graphmaker

Graphmaker notes:

Graph Maker (Excel) can be used to draw the graph for expression profile (6 ages; between 10 days – 20 weeks) of a gene of interest in pancreatic lymph nodes (PLN), spleen (SPL) and peripheral blood cells (PBC) in female NOD mice. The zero line represents the value of the gene expressed in the NOD.B10 tissue from pooled tissue samples collected at 21 days and 20 weeks (n=10 female NOD.B10 at both ages).

1. Select a gene of interest using the gene symbol or ID (e.g. ins2, NM_008387, A_51_P389597) from the IDD alphabet list (gene list).

2. Copy the entire row of the gene of interest.

3. Paste the row data to the 2nd row of the graph sheet.

4. The graphs of the gene expression profile in PLN, SPL and PBC will appear in the graph windows. Standard error bars for each gene in each tissue at each time represent the standard deviation (SD) of the replicate chip data for that oligo at that time in that tissue.

 

Investigators: Keiichi Kodama, Demi Dang, Mark Hartnett, Hideyuki Iiwai, Claire Holness, Atul Butte , and C. Garrison Fathman

 

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