Bovine serum (Invitrogen). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to

Bovine serum (Invitrogen). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were washed twice with PBS and total RNA was prepared by Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was reversed transcribed using dT15 and Superscript II (Invitrogen). PCR was run for 30 cycles at 94uC for 30 s, 60uC for 30 s, and 72uC for 30 s using Platinum Taq DNA polymerase (Invitrogen). PCR products were analyzed by agarose gel electrophoresis or DNA fragment analysis. For DNA fragment analysis, fluorescence 307538-42-7 web primer was used and products were mixed with size standard in formamide and analyzed on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). Data analysis was performed using GeneMapper software version 4.0. Primer sequences are summarized in Table 1.Materials and Methods Ethics StatementThe study was approved by the Ethics Committee of the Cross Cancer Institute and University of Alberta. Written informed consent was provided in accordance with the Declaration of Helsinki.Analysis of HAS1Vb/Vd Expression in Peripheral Blood Mononuclear Cells (PBMC)Peripheral blood samples were collected from normal individuals and MM patients at diagnosis. MM was identified based on consensus criteria. Normal blood was obtained from University of Alberta Hospital emergency room as anonymous samples from 102 individuals selected as being over the age of 50 and without any obvious hematological issues. PBMC were isolated by step gradient centrifugation (FicollPaque Plus; GE Healthcare). RT-PCR followed Transient expression and HAS1 splicing analysis SC-1 chemical information section, except that amplification was run for 35 cycles using E3/E5I4 primer set (Table 1) and PCR products were analyzed by DNA fragment analysis.Plasmid ConstructionHAS1FL (FLc) and HAS1g345 (G345) have been previously described [20,21]. In brief, FLc is generated by cloning of HAS1 cDNA fragment into a mammalian expression vector pcDNA3 (Invitrogen). G345 is generated by replacing exons 3-4-5 cDNA sequence in FLc with the corresponding genomic DNA fragment. Deletion constructs del5, del4, del3, del2 and del1 are derivatives of G345, being created by overlap extension PCR [22]. Two DNA subfragments were separately amplified: a) the 59 piece 23727046 extending from the beginning of the G345 construct to 680 bp downstream of exon 4, and b) the 39 piece extending from the selected sequence in intron 4 to the end of the G345 construct. Overlapping ends were created by primer design. Joining of fragments was performed by mixing equimolar ratio of DNA fragments in standard polymerase chain reaction (PCR) using HiFi Taq DNA polymerase (Invitrogen) in the absence of primers and run for 7 cycles at 94uC for 30 s and 72uC for 4 min. The assembled fragment was further amplified in the presence of forward and reverse primers for 30 cycles, and then cloned into pcDNA3. The end products are constructs that have selective internal intron 4 deletion, each carried 680 bp of upstream intronic sequence joined to a specified downstream intronic sequence. These are 489 bp (del5), 361 (del4), 263 bp (del3), 198 bp (del2) and 84 bp (del1) sequences upstream of exon 5. The deleted protions are calculated to be 983 bp, 1111 bp, 1209 bp, 1274 bp and 1388 bp respectively. Mutagenized HAS1 intron 3 (G1?8 m) was custom made by minigene synthesis (Mr.Gene). Constructs G345/G1?8 m and del1/G1?8 m were generated by o.Bovine serum (Invitrogen). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were washed twice with PBS and total RNA was prepared by Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was reversed transcribed using dT15 and Superscript II (Invitrogen). PCR was run for 30 cycles at 94uC for 30 s, 60uC for 30 s, and 72uC for 30 s using Platinum Taq DNA polymerase (Invitrogen). PCR products were analyzed by agarose gel electrophoresis or DNA fragment analysis. For DNA fragment analysis, fluorescence primer was used and products were mixed with size standard in formamide and analyzed on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). Data analysis was performed using GeneMapper software version 4.0. Primer sequences are summarized in Table 1.Materials and Methods Ethics StatementThe study was approved by the Ethics Committee of the Cross Cancer Institute and University of Alberta. Written informed consent was provided in accordance with the Declaration of Helsinki.Analysis of HAS1Vb/Vd Expression in Peripheral Blood Mononuclear Cells (PBMC)Peripheral blood samples were collected from normal individuals and MM patients at diagnosis. MM was identified based on consensus criteria. Normal blood was obtained from University of Alberta Hospital emergency room as anonymous samples from 102 individuals selected as being over the age of 50 and without any obvious hematological issues. PBMC were isolated by step gradient centrifugation (FicollPaque Plus; GE Healthcare). RT-PCR followed Transient expression and HAS1 splicing analysis section, except that amplification was run for 35 cycles using E3/E5I4 primer set (Table 1) and PCR products were analyzed by DNA fragment analysis.Plasmid ConstructionHAS1FL (FLc) and HAS1g345 (G345) have been previously described [20,21]. In brief, FLc is generated by cloning of HAS1 cDNA fragment into a mammalian expression vector pcDNA3 (Invitrogen). G345 is generated by replacing exons 3-4-5 cDNA sequence in FLc with the corresponding genomic DNA fragment. Deletion constructs del5, del4, del3, del2 and del1 are derivatives of G345, being created by overlap extension PCR [22]. Two DNA subfragments were separately amplified: a) the 59 piece 23727046 extending from the beginning of the G345 construct to 680 bp downstream of exon 4, and b) the 39 piece extending from the selected sequence in intron 4 to the end of the G345 construct. Overlapping ends were created by primer design. Joining of fragments was performed by mixing equimolar ratio of DNA fragments in standard polymerase chain reaction (PCR) using HiFi Taq DNA polymerase (Invitrogen) in the absence of primers and run for 7 cycles at 94uC for 30 s and 72uC for 4 min. The assembled fragment was further amplified in the presence of forward and reverse primers for 30 cycles, and then cloned into pcDNA3. The end products are constructs that have selective internal intron 4 deletion, each carried 680 bp of upstream intronic sequence joined to a specified downstream intronic sequence. These are 489 bp (del5), 361 (del4), 263 bp (del3), 198 bp (del2) and 84 bp (del1) sequences upstream of exon 5. The deleted protions are calculated to be 983 bp, 1111 bp, 1209 bp, 1274 bp and 1388 bp respectively. Mutagenized HAS1 intron 3 (G1?8 m) was custom made by minigene synthesis (Mr.Gene). Constructs G345/G1?8 m and del1/G1?8 m were generated by o.