VIEWS: 11 PAGES: 5 POSTED ON: 6/5/2010
Supplemental Materials & Methods RT-PCR primers nPTB exon 8 forward: 5′-GCATTTGCCAAGGAGACATCC nPTB exon 11 reverse: 5′-CGCTGCACATCTCCATAAACAC PTB exon 8 forward: 5′-AAGAGCAGAGACTACACTCGA PTB exon 12 reverse: 5′-CTGCCGTCTGCCATCTGCACAA src N1 exon forward: 5′-GGCCCTCTATGACTATGAG src N1 exon reverse: 5′-GCCAGCCACCAGTCTCCCTC Smap1_2 exon forward: 5′-AGCAGTAGAGTTTTTTCATCAGAGAT Smap1_2 exon reverse: 5′-GGCTTTTCTGGCTCCTTTTC Mtap2_2 exon forward: 5′-TCCCCAGCTACTCCTAAGCA Mtap2_2 exon reverse: 5′-GCCTGTGACGGATGTTCTTT CLCB EN exon forward: 5′-GGAGGAGTGGAACCAGCGCC CLCB EN exon reverse: 5′ –GGTCTCCTCCTTGGATTC Kif1b_3 exon forward: 5′-CGGTTCCACTGGTTCAAACT Kif1b_3 exon reverse: 5′-ACAGCCACACGAAGAAATCC Dst_2 forward: 5′-GATCTCGGTGGTAGCTCAGG Dst_2 reverse: 5′-TGTTTCCGGAGGAGAATGTC Dst_3 forward: 5′-AAGCACAGTGATGGTTCGTG Dst_3 reverse: 5′-TCCATGTTGGTCCTTCCTTT AA536749_5 forward: 5′-TCTAAAAGCAATCCTGACTTCCTG AA536749_5 reverse: 5′-GTGTCAGTGGAATGGATGCAGC Mybl1_2 forward: 5′-CACTCTGCATCTGTGAAGAAG Mybl1_2 reverse: 5′-TGTCTTCCCATATACCACTGTTTC Chr5.809_1 forward: 5′-TGGCACCTCTAAAGAAAGCAA Chr5.809_1 reverse: 5′-ACATGTTCCCCATGTCCAGT Atp2b1_1 forward: 5′-GTGGCCAGATCTTGTGGTTT Atp2b1_1 reverse: 5′-CATCAATAAGGGGGATGTGC Ktn1_2 forward: 5′-TAGAGAAGGCCGAGATGGAG Ktn1_2 reverse: 5′-CCTTCTTTCTCTCCGTTTGTTC Real-time PCR primers ACTB forward: 5′-TACAGCTTCACCACCACAGC ACTB reverse: 5′-ATGCCACAGGATTCCATACC GAPDH forward: 5′- GAPDH reverse 5′- PTB forward: 5′-TCTACCCAGTGACCCTGGAC PTB reverse: 5′-GAGCTTGGAGAAGTCGATGC nPTB forward: 5′-ACCAGGCATTTTTGGAACTG nPTB reverse 5′-TGTGGTGCCACTAAGAGGTG siRNA template oligonucleotides pRL156(luciferase siRNA), sense: 5′-AATAAATAAGAAGAGGCCGCGCCTGTCTC pRL156(luciferase siRNA), antisense: 5′-AACGCGGCCTCTTCTTATTTACCTGTCTC Upf1 siRNA, sense: 5′-AATTCAGTTTTAGCAGTGGAACCTGTCTC Upf1 siRNA, antisense: 5′-TTTTCCACTGCTAAAACTGAACCTGTCTC shRNA construct oligonucleotides si-m-PTB-forward 5′-GAGAGAAGACTAAAAATGACTTACAGACCAGACATTATAAGAGTAA si-m-PTB-reverse 5′-GAGAGAAGACTAAAAATGACTTACAGACCAGAGATTACTCTTATAA si-nPTB-v3-forward 5′-GAGAGAAGACTAAAAATGGTCTAGATTCAGTTATGAATAAGAGTTC si-nPTB-v3-reverse 5′-GAGAGAAGACTAAAAATGGTCTAGATTCAGTTATGAACTCTTATTC Construction of nPTB exon 10 minigenes The primer nPTB-E10MG-F2A_Bgl was used for both minigenes B and C. The PCR products were cloned using ApaI and BglII sites, replacing the middle exon of Dup4-1 in which the constitutive exons and intervening intron sequences are derived from β-globin (Modafferi, E.F. and D.L. Black. A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon. (1997). Molec. and Cell. Biol. 17(11):6537-6545). Minigene A: nPTB_E10MG-F1_Apa: 5’-aaatgggcccATTCTGCGGGAACCACCCTTCGTTATG nPTB_E10MG-R2_Bgl: 5’- aaaragatctGCCAAAGTGCTAGCTACAAATAAGAA Minigene B: nPTB-E10MG-F2A_Bgl: 5’- attagggcccATTTTCTGACCAAATTCCTGCATTTCCTATGTACTGACC nPTB_E10MG-R1_Bgl: 5’- aaatagatctATAACATAccgaagagggtaaacaga Minigene C: nPTB-E10MG-F2A_Bgl: 5’-attagggcccATTTTCTGACCAAATTCCTGCATTTCCTATGTACTGACC nPTB_E10MG-R3A_Bgl: 5’- Aaatagatctaaagaccatgcaaaataaattagcatgca Splicing Microarray Design and Construction of microarray Exons for the array were selected based on the criteria presented in the Materials and Methods section. The exon sets were combined, filtered for redundancies and used to build models for the corresponding transcripts. Oligonucleotide probes for the exon-exon junctions from skipped and included isoforms were designed, along with probes for sequence within the alternative exon itself and within adjacent constitutive exons. Oligonucleotides were spotted in duplicate. Briefly, 5' amino-linker containing oligonucleotides at 33uM in print buffer (150 mM NaPO4, 6.25% Na2SO4, 0.0005% SDS) were printed and covalently attached to Codelink slides (GE). Printing was done on a linear servo-driven robotic contact pin printer essentially as described by the DeRisi lab at UCSF (http://derisilab.ucsf.edu/arraymaker.shtml). Coupling and post-processing of slides were described previously (Clark et al., 2002). cDNA labeling and microarray hybridization 5 to 10µg of poly-A selected RNA were annealed to 450µmol mix of random hexamers and dT VN. The RNA was then reverse transcribed in the presence of 300µM amino- 24 allyl dUTP (Ambion), 100µM dTTP and 500µM each dATP, dCTP and dGTP. The reaction was incubated for 2 hours at 48ºC and stopped by the addition of NaOH and EDTA to final concentration of 100mM and 2mM respectively. The RNA was hydrolyzed by incubation at 65ºC for 20 minutes and the solution was neutralized by the addition of HEPES pH7.5 to 500µM final concentration. The cDNA was purified using DNA clean and concentrator kit (Zymo research) and eluted in 50mM NaHCO buffer 3 (pH9.0). The amino-allyl labeled cDNA was coupled to either Cy3 or Cy5 ester (GE) according to the manufacturers protocol. The fluorescently labeled cDNA was purified using DNA clean and concentrator kit and eluted in 14µl of 10mM Tris buffer pH8.0. The hybridization mix was prepared by combining 10µl of Cy3 and Cy5 labeled samples and adding 0.66µl 1M HEPES (pH7.5), 1.65µl poly-A (2µg/µl), 6.6µl 20xSSC and 3.3µl 1% SDS. The mix was denatured at 95ºC for 3 minutes, cooled down and applied to the microarray slide. The hybridization was carried out at 63ºC for 16 to 20 hours. The slides were then rinsed with 4XSSC/0.1% SDS prewarmed to 60C and washed twice for 5 minutes in 2xSSC/0.1% SDS prewarmed to 60ºC, and once in each 0.2xSSC and 0.05xSSC for 1 minute at room temperature. The slides were dried by centrifugation at 800rpm for 4 minutes and scanned immediately on a GenePix 4000b scanner (Molecular Devices). During the prescan, the photomultipliers were adjusted to avoid saturation and to balance the signal of the two channels. Microarray image processing, array normalization and data visualization Scanned image data were analyzed using GenePix Pro6 software (Axon) as described previously (Srinivasan et al., 2005). Briefly, each spot was defined within a grid layout over the image. Flagging criteria were applied to exclude bad spots as detailed (Srinivasan et al., 2005). For each channel, the median values of the qualified spots were determined after subtraction of local background intensities. The result files generated from the GenePix pro 6 were then analyzed in R using Bioconductor. Spots with intensities lower than the negative control spots were excluded from the analysis. Global normalization was then performed using print-tip lowess method. For each gene, the probes were categorized as constitutive, inclusion, or exclusion probes according to their splicing models. To adjust for changes in overall expression, we performed “within-gene normalization” by subtracting the average log2 ratio of the constitutive probes from the log2 ratios of every individual alternative probe. To identify significant differences in exon splicing, we performed two sample t-tests between the log2 ratios of the inclusion probe set and the exclusion probe set, and calculated p-values for every alternative exon. Alternative exons with a non-zero difference in log ratio between inclusion and exclusion probes at a p-value of less than 0.05 were considered to have splicing changes. Alternative exons with p-values lower than 0.05 were subjected to resampling-based false discovery rate analysis (FDR), to control for the multiplicity and ranked by their FDR value.
Pages to are hidden for
"RT-PCR Primers"Please download to view full document