Total RNA extracted using Trizol following manufacturer's instructions. The procedures for multiplex amplification and genotype determination are illustrated in Figure 1. First, multiplex PCR (Fig. 1A) was performed in 30 µL of PCR mix containing 1x PCR buffer (50 mM KCl, 100 mM Tris-HCl at pH 8.3, 1.5 mM MgCl2, and 100 µg/mL gelatin), four dNTPs (200 µM each; Invitrogen), primers (20 nM each) for all SNPs in the multiplex group, 6 units of HotStar Taq DNA polymerase (QIAGEN), and 5 ng of DNA or single sperm. The samples were first heated to 94°C for 15 min to activate the Taq DNA polymerase followed by 40 PCR cycles. Each PCR cycle consisted of 40 sec at 94°C for denaturation and 2 min at 55°C followed by 5 min of ramping from 55°C to 70°C for annealing and extension. A final extension step was carried out at 72°C for 3 min at the end of the 40-th cycle. PCR amplifications were performed with thermal cyclers capable of ramping as slow as 0.01°C/sec. ssDNA was generated in both directions by using the same conditions for multiplex PCR except: (1) 1-2 µL of product from the multiplex PCR was used as templates, (2) only one primer for each SNP; and (3) 45 PCR cycles.
Label
Cy5-ddCTP
Label protocol
Probes were labeled in 25 µL of labeling solution containing Sequenase buffer), 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°C for 10 min. The slide was washed under conditions as described above.
Total RNA extracted using Trizol following manufacturer's instructions. The procedures for multiplex amplification and genotype determination are illustrated in Figure 1. First, multiplex PCR (Fig. 1A) was performed in 30 µL of PCR mix containing 1x PCR buffer (50 mM KCl, 100 mM Tris-HCl at pH 8.3, 1.5 mM MgCl2, and 100 µg/mL gelatin), four dNTPs (200 µM each; Invitrogen), primers (20 nM each) for all SNPs in the multiplex group, 6 units of HotStar Taq DNA polymerase (QIAGEN), and 5 ng of DNA or single sperm. The samples were first heated to 94°C for 15 min to activate the Taq DNA polymerase followed by 40 PCR cycles. Each PCR cycle consisted of 40 sec at 94°C for denaturation and 2 min at 55°C followed by 5 min of ramping from 55°C to 70°C for annealing and extension. A final extension step was carried out at 72°C for 3 min at the end of the 40-th cycle. PCR amplifications were performed with thermal cyclers capable of ramping as slow as 0.01°C/sec. ssDNA was generated in both directions by using the same conditions for multiplex PCR except: (1) 1-2 µL of product from the multiplex PCR was used as templates, (2) only one primer for each SNP; and (3) 45 PCR cycles.
Label
Cy3-ddUTP
Label protocol
Probes were labeled in 25 µL of labeling solution containing Sequenase buffer), 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°C for 10 min. The slide was washed under conditions as described above.
Hybridization protocol
Hybridization was done with 1x hybridization solution (5x Denhart's solution, 0.5% SDS, 5x SSC, 20 µL of ssDNA/1000 microarray spots) in a Hybridization Chamber (Corning) at 56°C for 2.5-4 h. After hybridization, the slide was washed at 56°C with 1x SSC and 0.1% SDS for 10 min, twice with 0.5x SSC for 30 sec, and twice with 0.2x SSC for 30 sec.
Scan protocol
Microarrays were scanned with GenePix 4000B (Axon Instruments)
Description
#012_C4ag_R590G490_50320.gpr Single sperm from donor#12
Data processing
The resulting images were analyzed with either the GenePix Pro (Axon Instruments) or ImaGene (BioDiscovery) software.
