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(1) Gene expression profiling of neurofibromas
The GMRCL Human 10K set, Version 1 chips which containing 11,000 sequence-verified human genes selected from InCyte were used in this study. Fabrication of the slides, hybridization, washing and detection of signals were carried out in the Genome Medical Research core laboratory of Chang Gung Memorial Hospital as described previously [12]. An indirect labelling of cDNA targets using the 3DNA Sunmicro EX Expression Array Detection kit (Genisphere, Penn, USA) were applied on 2 μg of total RNA isolated from each experimental sample.? After hybridization and washing, slides were scanned with a confocal scanner ChipReader (Virtek, Canada). The spot and background intensities were then acquired with GenePix Pro 4.1 software (Axon Instruments, Inc., Calif, USA), and a pre-analysis data management, flooring and within-slide normalization, was performed using MATLAB 6.0 software (The MathWorks, Inc., Mass, USA). We averaged the log ratios of the duplicated spots on each slide in dye swapping experiments. The differential expression was considered significant when the average ratio of the signal between the cancer and normal tissue specimens was greater than 2.? Detailed information in MIAME format, including all raw microarray data, can be found at http://www.cgmh.org.tw/intr/intr2/c32a0/index.htm/. Of 22092 cDNA fragments detected, 1435 genes showed over- and under- expressed more than two-fold compared with normal nerve tissue in more than 50% of the neurofibromas analyzed. These genes are mostly related to metabolism, cell growth, cell differentiation, immunity, and protein modulation.
To confirm the expression profile of these genes in neurofibromas, laboratory-based validation of data has been used in our study. Real-time RT-PCR which can quantitatively measuring specific mRNAs is our first choice. This method is rapid, relatively inexpensive and requires minimal starting template. However, it requires a significant up-front effort to optimize amplification conditions, and the method has potential pitfalls that must be carefully monitored. As a proof-of-principle and to verify our microarray data, we have selected 10 genes and examine their expression levels in a panel of tumors, cell lines and normal samples by means of real-time quantitative PCR (TaqMan real-time PCR machine, ABI 7700 Sequence Detector, Applied Biosystems). Each single-stranded cDNA has been reverse-transcribed from total RNA and diluted for subsequent PCR amplification. The PCR reaction will be normalized to the ribosomal protein L11 (RPL11) level. Each PCR will be carried out in a 25ul volume reaction containing 12.5ul TaqMan Universal MasterMix (Applied Biosystems), 0.25ul of each forward and reverse primers (10 uM), 2.0ul of dual-labeled probe (2.5uM), and cDNA from 50ng total RNA.? Each reaction will be amplified by 40 cycles of 95 oC for 15 sec and 60 oC for 1 min.? The primers and probes of selected genes or sequences used for real-time quantitative PCR has been obtained from GeneBank and placed into primer design software (PrimerExpress version 1.5, Applied Biosystems). This experiment is still in processing.
(2) Detection of large gene alterations
Analysis of LOH (Loss of Heterozygosity) and microsatellite instability
The allelic loss analysis and microsatellite instability were sought by typing genomic DNA from paired tumor and/or lymphocyte samples with four microsatellite markers located within the NF1 gene (IVS27AAAT2.1, IVS27CA28.4, IVS27TG24.8, IVS38GT53). All these primers will be labeled with fluorescence dye fluorescein (FAM) or rhodamin (Tamra) at the 5’-terminus.? Amplified markers (0.5ul each) from each sample were pooled together and analyzed under denaturing conditions using an ABI Genetic Analyzer 3700 (Applied Biosystems).
Affymetrix Human U133A 10K CGH
The 10K SNP array provides comprehensive coverage of the genome for genotyping studies. Each array contained 11,555 biallelic polymorphic sequence randomly distributed throughout the genome, except for the Y chromosome.? The median physical distance between SNPs is ~105 kb and the mean distance between SNPs is 210 kb. The average heterozygosity for these SNPs is 0.37, with an average minor allele frequency of 0.25.? The algorithm used for making genotype calls was described previously by Affymetrix.? A total of 250ng germline DNA was digested with XbaI and then ligated to XbaI adaptor before subsequent PCR amplification.? All the steps mentioned above were carried on in the pre-PCR clean room.? Cycling was conducted as follows: 95℃ for 3 minutes followed by 35 cycles for 20 seconds, 59℃ for 15 seconds, and 72℃ for 15 seconds.? Final extension was done at 72℃ for 7 minutes.? To evaluate PCR products, 3 ul of each PCR product was mixed with 3 ul of the 2 x gel loading dye on 2% Tris-borate EDTA gel and run at 120 V for 1 hour to check for the expected product between 250 and 1 kb. After purification and elution of the PCR products using Qiagen MinElute 96, quantification of purified PCR product was done using spectrophotometric analysis. A final 20 ug of PCR product was fragmented with DNase I.Successful fragmentation was confirmed by the presence of a smear with the darkest region corresponding to 50 to 100 bp. The fragmented PCR product was end labeled with biotin and hybridized to the array.? Arrays were incubated at 48℃ for 48 hours in the Affymetrix GeneChip systemhybridization oven. Microarrays were washed and stained in the Gene Chip Fluidics Station 450 (Affymetrix) following the manufacturer’s instructions.
Date |
Case |
Chromosomal changes |
12/2/2005 |
Yang T |
17 del |
1/19/2006 |
Lee PB |
17 del |
1/19/2006 |
Lee PT |
17 del |
1/19/2006 |
Chou T |
17 del |
1/19/2006 |
Lee DB |
Normal |
1/19/2006 |
Lee DT |
17 del |
2/27/2006 |
Hung B |
Normal |
3/9/2006 |
Hung T |
Failed |
3/9/2006 |
Huang B |
Normal |
(3) Identifying mutations at GAP-related domain (GRD) using SSCP analysis.
Genomic DNA polymerase chain reaction were carried out in 50ul reaction volumes containing 50ng genomic DNA, 0.2 M of each forward and reverse primer, 0.1 mM of each dNTP, 5 �愮 10X PCR buffer ( 100mM TRIS pH 8.3, 500mM KCl, 15mM MgCl2, 0.01% gelatin) and 2.5 units AmpliTaq polymerase (Applied Biosystems) with the following amplification condition: an initial 10 min denaturation at 95℃ followed by 40 cycles of denaturation at 95℃ for 30 sec, specific annealing temperature for 30 sec, extension at 72℃ for 30 sec and followed by a 10 min final extension at 72℃.? All thermal cycles were run on a Applied Biosystem 9600 thermocycler.? PCR products were then screened for mutations by single-strand conformational polymorphism (SSCP) analysis using precasted GeneGel Excel 12.5/24 acrylamide gels (Amercham-Pharmacia). For SSCP, six microliters of denatured PCR product were mixed with loading buffer and loaded into the gels (Pharmacia). ?Electrophoresis typically was performed at 15℃ with 600 V for 2 hrs. ?After electrophoresis, the gels were silver stained using DNA Silver Staining Kit (Phamacia Biotech).? All detected SSCP variants were then be confirmed by DNA sequencing (ABI 3700 automatic sequencer, PE Applied Biosystems).
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