Of AmpliTaq Gold DNA Polymerase (Applied Biosystems). PCR was conducted under the following cycling conditions: a pre-PCR incubation step at 95uC for 15 min; followed by 35 cycles of 95uC for 15 s, 55uC for 45 s, and 72uC for 30 s; and a final extension of 72uC for 10 min. The amplified fragments were separated in 6 denaturing polyacrylamide gels on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems), as described in the manufacturer’s instructions. Normal and tumor DNA pairs were compared for changes in the number of allele peaks and the peak height of each marker by using GeneScan Analysis software (Applied Biosystems). The LOH index of each normal and tumor DNA pair was calculated as previously described [16]. Briefly, the ratio of the allele peak heights calculated for each tumor sample was divided by the allele peak height ratio of the normal matching control. An LOH index of #0.67 or 1.5, representing at least a 33 decrease of a tumor allele, was indicative of allelic loss.Data are n ( ), unless otherwise noted. Pearson Chi-square test, unless otherwise noted. c Student’s t-test. d Linear-by-linear association chi-square test. e Only Dukes’ stages B and C were observed. doi:10.1371/journal.pone.0067040.tbto the manufacturer’s instructions. The concentration and purity of RNA were determined with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific), and RNA integrity was confirmed by agarose gel electrophoresis.RNA ExtractionTotal RNA was extracted from the frozen tissues and 10 CRC cell lines (COLO205, HCC2998, HCT116, HCT15, HT29, KM12 and SW620 from the US National Cancer Institute; LoVo, SW48, and SW480 from the Bioresource Collection and Research Center, Taiwan) by using TRIzol reagent (Invitrogen) accordingReverse Transcription-Polymerase Chain Reaction (RTPCR)Ten randomly selected CRC cases were used in a pilot study for gene expression. Complementary DNA (cDNA) was reversetranscribed from total RNA (2 mg/20 mL reaction) by using the High Capacity cDNA Reverse Title Loaded From File transcription Kit (AppliedGenetic Loss of NDST4 in Colorectal CancerFigure 1. NDST4 is identified as the candidate CRC-associated tumor suppressor gene at chromosome 4q26. A. Title Loaded From File Microsatellite markers used for loss of heterozygosity study. Three genes are located in the minimal deletion region delineated by D4S2297 and D4S2303. Black bars indicate UGT8 and NDST4 genes. miR-577 (MIR577) lies in the intron of UGT8. B. Analysis of UGT8 and NDST4 mRNAs in tumors (T) and matched normal mucosae (N) of CRC tissues by RT-PCR. b-ACTIN was used as an internal RNA control. C. Analysis of miR-577 expression in CRC tissues by qRTPCR. The expression levels of tumors were normalized to those of corresponding normal mucosae. Data represent the mean 6 SD. doi:10.1371/journal.pone.0067040.gBiosystems). Reverse transcription was conducted under the following conditions: 25uC for 10 min, 37uC for 2 h, and 85uC for 5 min. The resultant cDNA was diluted 5-fold with diethylpyrocarbonate (DEPC)-treated H2O. Gene-specific primer sets designed spanning exons were as follows: NDST4 forward 59TCTGGGAGTTACACCTCG-39 and reverse 59-TCTTGAGAGGCTTAGTTCTTG-39; UGT8 forward 59-TTATATTATTCGTCACAATGG-39 and reverse 59-AAAACTAAGGTCTGACACAGT-39; b-ACTIN forward 59ACAGAGCCTCGCCTTTGC-39 and reverse 59TCATCTTCTCGCGGTTGG -39. PCR amplification was conducted in a final volume of 25 mL by using 2.5 mL of diluted cDNA, 1 mM of each of respective primers, 250 mM of each dNTP, and 1 unit of Super-Therm Gold.Of AmpliTaq Gold DNA Polymerase (Applied Biosystems). PCR was conducted under the following cycling conditions: a pre-PCR incubation step at 95uC for 15 min; followed by 35 cycles of 95uC for 15 s, 55uC for 45 s, and 72uC for 30 s; and a final extension of 72uC for 10 min. The amplified fragments were separated in 6 denaturing polyacrylamide gels on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems), as described in the manufacturer’s instructions. Normal and tumor DNA pairs were compared for changes in the number of allele peaks and the peak height of each marker by using GeneScan Analysis software (Applied Biosystems). The LOH index of each normal and tumor DNA pair was calculated as previously described [16]. Briefly, the ratio of the allele peak heights calculated for each tumor sample was divided by the allele peak height ratio of the normal matching control. An LOH index of #0.67 or 1.5, representing at least a 33 decrease of a tumor allele, was indicative of allelic loss.Data are n ( ), unless otherwise noted. Pearson Chi-square test, unless otherwise noted. c Student’s t-test. d Linear-by-linear association chi-square test. e Only Dukes’ stages B and C were observed. doi:10.1371/journal.pone.0067040.tbto the manufacturer’s instructions. The concentration and purity of RNA were determined with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific), and RNA integrity was confirmed by agarose gel electrophoresis.RNA ExtractionTotal RNA was extracted from the frozen tissues and 10 CRC cell lines (COLO205, HCC2998, HCT116, HCT15, HT29, KM12 and SW620 from the US National Cancer Institute; LoVo, SW48, and SW480 from the Bioresource Collection and Research Center, Taiwan) by using TRIzol reagent (Invitrogen) accordingReverse Transcription-Polymerase Chain Reaction (RTPCR)Ten randomly selected CRC cases were used in a pilot study for gene expression. Complementary DNA (cDNA) was reversetranscribed from total RNA (2 mg/20 mL reaction) by using the High Capacity cDNA Reverse Transcription Kit (AppliedGenetic Loss of NDST4 in Colorectal CancerFigure 1. NDST4 is identified as the candidate CRC-associated tumor suppressor gene at chromosome 4q26. A. Microsatellite markers used for loss of heterozygosity study. Three genes are located in the minimal deletion region delineated by D4S2297 and D4S2303. Black bars indicate UGT8 and NDST4 genes. miR-577 (MIR577) lies in the intron of UGT8. B. Analysis of UGT8 and NDST4 mRNAs in tumors (T) and matched normal mucosae (N) of CRC tissues by RT-PCR. b-ACTIN was used as an internal RNA control. C. Analysis of miR-577 expression in CRC tissues by qRTPCR. The expression levels of tumors were normalized to those of corresponding normal mucosae. Data represent the mean 6 SD. doi:10.1371/journal.pone.0067040.gBiosystems). Reverse transcription was conducted under the following conditions: 25uC for 10 min, 37uC for 2 h, and 85uC for 5 min. The resultant cDNA was diluted 5-fold with diethylpyrocarbonate (DEPC)-treated H2O. Gene-specific primer sets designed spanning exons were as follows: NDST4 forward 59TCTGGGAGTTACACCTCG-39 and reverse 59-TCTTGAGAGGCTTAGTTCTTG-39; UGT8 forward 59-TTATATTATTCGTCACAATGG-39 and reverse 59-AAAACTAAGGTCTGACACAGT-39; b-ACTIN forward 59ACAGAGCCTCGCCTTTGC-39 and reverse 59TCATCTTCTCGCGGTTGG -39. PCR amplification was conducted in a final volume of 25 mL by using 2.5 mL of diluted cDNA, 1 mM of each of respective primers, 250 mM of each dNTP, and 1 unit of Super-Therm Gold.
