The fresh tissues were immediately immersed in RNAlater (Ambion, Inc., USA) after surgical resection, stored at 4℃ overnight to allow thorough penetration of the RNase inhibitor, and then frozen at -80℃ until use.
Extracted molecule
genomic DNA
Extraction protocol
After total RNA extraction using TRIzol reagent (Invitrogen, USA), DNA in the interphase and phenol phase from the initial homogenate was isolated according to the manufacturer's instructions.
Label
Cy5 [Cy5-ddGTP/Cy5-ddCTP]
Label protocol
Labeling probes (NOT labeling DNA, detail infromation can be found in Genome Res. 2005 Feb;15(2):276-83.) by single-base extension. The 3'ends of the oligonucleotide probes hybridizing to the ssDNA were immediately next to the polymorphic sites. Using ssDNA as a template,each probe was extended by a single dideoxynucleoside triphosphate (ddNTP) conjugated to a fluorescent chromophore (Cy3 or Cy5). In this way, probes hybridizing to different allelic sequences were labeled with different fluorescent colors. Theoretically, when hybridizing ssDNA contains a homozygous SNP, a probe should predominantly incorporate one color (signal color that is specific) over the other (background color),while a probe hybridizing to ssDNA containing a heterozygous SNP should incorporate both colors equally. After normalization and background subtraction, the genotypes were determined based on the logratios between the two normalized color intensities by using empirical linear values as cutoffs, which divided SNPs into three groups, two homozygous and one heterozygous. The cutoff values were validated by comparing the microarray results with those obtained by using independent genotyping methods. The following is the detail for labeling experiment. Probes were labeled in 25μL of labeling solution containing 1/7 volume of Sequenase buffer (supplied by the vendor),0.5 units/μL Sequenase (Amersham Pharmacia Biosciences), Cy3-ddATP and Cy5-ddGTP (PE Biosystems) for AG probes,and Cy3-ddUTP and Cy5-ddCTP for CT probes (750 nM each). The reaction was incubated at 70℃ for 10 min.
The fresh tissues were immediately immersed in RNAlater (Ambion, Inc., USA) after surgical resection, stored at 4℃ overnight to allow thorough penetration of the RNase inhibitor, and then frozen at -80℃ until use.
Extracted molecule
genomic DNA
Extraction protocol
After total RNA extraction using TRIzol reagent (Invitrogen, USA), DNA in the interphase and phenol phase from the initial homogenate was isolated according to the manufacturer's instructions.
Label
Cy3 [Cy3-ddATP/Cy3-ddUTP]
Label protocol
Labeling probes (NOT labeling DNA, detail infromation can be found in Genome Res. 2005 Feb;15(2):276-83.) by single-base extension. The 3'ends of the oligonucleotide probes hybridizing to the ssDNA were immediately next to the polymorphic sites. Using ssDNA as a template,each probe was extended by a single dideoxynucleoside triphosphate (ddNTP) conjugated to a fluorescent chromophore (Cy3 or Cy5). In this way, probes hybridizing to different allelic sequences were labeled with different fluorescent colors. Theoretically, when hybridizing ssDNA contains a homozygous SNP, a probe should predominantly incorporate one color (signal color that is specific) over the other (background color),while a probe hybridizing to ssDNA containing a heterozygous SNP should incorporate both colors equally. After normalization and background subtraction, the genotypes were determined based on the logratios between the two normalized color intensities by using empirical linear values as cutoffs, which divided SNPs into three groups, two homozygous and one heterozygous. The cutoff values were validated by comparing the microarray results with those obtained by using independent genotyping methods. The following is the detail for labeling experiment. Probes were labeled in 25μL of labeling solution containing 1/7 volume of Sequenase buffer (supplied by the vendor),0.5 units/μL Sequenase (Amersham Pharmacia Biosciences), Cy3-ddATP and Cy5-ddGTP (PE Biosystems) for AG probes,and Cy3-ddUTP and Cy5-ddCTP for CT probes (750 nM each). The reaction was incubated at 70℃ for 10 min.
Hybridization protocol
Hybridization was done with 1 X hybridization solution (5 X Denhart's solution, 0.5% SDS,5 X SSC,20μL of ssDNA/1000 microarray spots) in a Hybridization Chamber (Corning) at 56℃ for 2.5-4h. Chambers were emerged in iced water for 30 sec before opening. The slide was washed at 56℃ with 1 X SSC and 0.1% SDS for 10min,twice with 0.5 X SSC for 30sec, and twice with 0.2 X SSC for 30 sec.
Scan protocol
The microarray was scanned by a LuxScanTM-10K Scanner (CapitalBio Inc., Beijing,China).
Data processing
Genotype call for each SNP was determined by a computer program called AccuTyping2b (Nucleic Acids Res 2006;34: e116) based on two color signal intensities. If the log ratio of the two color (Cy5 and Cy3) intensities for a SNP was between -3.3 and 3.3, the SNP genotype was considered as heterozygous (C/T). If the log ratio was greater than 3.3 or less than -3.3, the genotype was assigned as homozygous, C/C and T/T, respectively.
