Microdissected sections of fresh fixed paraffin embedded tissue
Extracted molecule
genomic DNA
Extraction protocol
DNA extracted using a QIAGEN DNA Mini Kit (QUIAGEN Inc., Canada) after a 72 hr period in lysis buffer at 56°C. DNA from 12 normal lymph nodes was prepared similarly and pooled for the reference sample. The single cell comparative genomic hybridization protocol (SCOMP) was used for genome amplification
Label
Cy5
Label protocol
1.0μg of amplified test and reference DNA was mixed with 21.5μl ddH2O and 20μl of 2.5X Random Primer/Reaction Mix Buffer (Invitrogen, www.invitrogen.com), boiled for 5 min. and 5μl of ‘low C’ dNTP mix with either 2.5μl Cy5-dCTP (Amersham Biosciences, USA) for test or Cy3 for the reference DNA with 1μl of Klenow solution, then incubated at 37°C for 2 hrs. The reaction was stopped by adding EDTA, then 450μl of TE pH 7.4 added and the mix loaded onto a micron-30 filter (Fisher/Millipore), spun for 8-10 min. at 10,000 rpm, inverted and spun for 1 min to recover the purified probe.
Microdissected sections of fresh fixed paraffin embedded tissue
Extracted molecule
genomic DNA
Extraction protocol
DNA extracted using a QIAGEN DNA Mini Kit (QUIAGEN Inc., Canada) after a 72 hr period in lysis buffer at 56°C. DNA from 12 normal lymph nodes was prepared similarly and pooled for the reference sample. The single cell comparative genomic hybridization protocol (SCOMP) was used for genome amplification
Label
Cy3
Label protocol
1.0μg of amplified test and reference DNA was mixed with 21.5μl ddH2O and 20μl of 2.5X Random Primer/Reaction Mix Buffer (Invitrogen, www.invitrogen.com), boiled for 5 min. and 5μl of ‘low C’ dNTP mix with either 2.5μl Cy5-dCTP (Amersham Biosciences, USA) for test or Cy3 for the reference DNA with 1μl of Klenow solution, then incubated at 37°C for 2 hrs. The reaction was stopped by adding EDTA, then 450μl of TE pH 7.4 added and the mix loaded onto a micron-30 filter (Fisher/Millipore), spun for 8-10 min. at 10,000 rpm, inverted and spun for 1 min to recover the purified probe.
Hybridization protocol
Labeled products were combined, 80μg of Human Cot-1 DNA and 200mg of yeast tRNA (Invitrogen) were added. The mixture was concentrated in a speed-vac at 37°C down to 10μl and resuspended in 80μl of DIG Easy Hybe buffer (Roche, Switzerland), denatured at 100°C for 1.5 min., cooled on ice and incubated for 30 min at 37°C, then hybridized to 19K human cDNA arrays (University Health Network Microarray Centre, Toronto, Canada, www.microarrays.ca) for 16 hours at 42°C.
Scan protocol
GenePix 4000B slide scanner and GenePix Pro 6.0 software package (Molecular Devices, California, USA)
Description
run in duplicate
Data processing
Samples run in duplicate, Log2 ratios of duplicates averaged, filtered to exclude cDNA clones without mapping info and those spots flagged as anomalous, cenetered by the median and scale normalized.
