Gene expression profiles in different proglottids of Moniezia expansa

We used complementary DNA microarray to analyze the gene expression profiles in different proglottids of Moniezia expansa. A total of 4056 sequences including full length and partial complementary DNAs representing novel, known, and control genes were analyzed. We utilized cDNA array for detection of gene expression profiles of different proglottids of M.expansa. The present study provides some interesting data to better understanding the mechanisms of procreation and may suggest some potential target molecules for a more effective treatment on verminosis. 1.Construction of microarray By clustering the 2,642 sequences from the cDNA library, 1,078 unigenes of M.expansa, including full-length and partial cDNAs representing novel or known genes, were obtained for array construction. The negative control spots of non-tapeworm origin in the chip were the rice genes (48 spots). In addition, the array included 9 known genes(54 spots) as positive control genes that were provided by our library and 24 empty spots. Unigenes and all control genes were cloned into plasmid vector. The DZH (TC) chips were constructed by United Gene Holdings Limited of China (Shanghai, China) according to the method described by Li et al. The cDNA inserts were amplified by use of the polymerase chain reaction (PCR) using universal primers to plasmid vector sequences and were then purified. All PCR products were examined by agarose gel electrophoresis to ensure the quality and the identity of the amplified clones as expected. Then the amplified PCR products were dissolved in a buffer containing 3×SSC solution. The solution with amplified PCR products were spotted onto glass slides (model DZH-TC) using a Cartesian PixSys 7500 motion control robot (Cartesian Technologies, Irvine, CA., USA) fitted with ChipMaker Micro-Spotting Technology (TeleChem International, Sunnyvale, CA., USA). The glass slides were then hydrated for 2 h in 70% humidity, dried for 0.5 h at room temperature, and UV crosslinked (65 mj/cm). They were further processed at room temperature by soaking in 0.2% sodium dodecyl sulfate (SDS) for 10 min, distilled H2O for 10 min, and 0.2% sodium borohydride (NaBH4) for 10 min. The slides were dried again and ready for use. 2.RNA preparation and probe labeling Total RNAs were isolated from 6 M.expansas with scolex-neck proglottids, immature proglottids, mature proglottids and gravid proglottids. Tissue samples were ground into a fine powder in a 10 cm ceramic mortar (RNase-free) and were then homogenized in TRIzol (Biostar, Shanghai, China). After centrifugation, the supernatant was separated from the organic phase and was extracted in an equal volume of chloroform. The aqueous phase was then precipitated by an equal volume of isopropanol at 4°C, centrifuged to pellet the RNA and dissolved in Milli-Q H2O. The fluorescent cDNA probes were prepared through reverse transcription with Cy3- or Cy5-deoxy UTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as follows: 5 μg of oligo(dT18) was added and annealed to 3μg of mRNA by heating the mixture to 70˚C for 10 min, and then chilling it on ice. The final reaction buffer mixture contained dNTPs (200 μM dATP, dCTP and dGTP; 60 μM dTTP; and 60 μM Cy3- or Cy5-dUTP), 2 μl of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 1×reaction buffer 10μl. The reactions were carried out at 42 ºC for 2 h. RNA was hydrolyzed by adding 4 μl of 2.5 M NaOH, incubating the mixture at 65 ºC for 10 min and then neutralizing it with 4 μl of 2.5 M HCl. The RNA samples from the scolex-neck proglottids were labeled with Cy3-dUTP and those from immature proglottids, mature proglottids and gravid proglottids, respectively, were labeled with Cy5-dUTP. The two color probes were then mixed and diluted to 500 μl with TE, and concentrated using a Microcon YM-30 filter (Millipore, Bedford, MA, USA) to 10 μl. The sample was then vacuum dried. 3.Hybridization The probe was dissolved in 20 ml of hybridization solution (5×SSC (0.75M NaCl and 0.075M sodium citrate), 0.4% SDS, 50% formamide). Microarrays were pre-hybridized with a hybridization solution containing 0.5 mg/ml of denatured salmon sperm DNA at 42°C for 6 h. Fluorescent probe mixtures were denatured at 95°C for 5 min and then applied onto the pre-hybridized chip under a cover glass. Chips were hybridized at 42°C for 15-17 h. Next, the hybridized chips were each washed at 60°C for 10 min in solutions of 2×SSC and 0.2% SDS, 0.1×SSC and 0.2% SDS, 0.1×SSC and then dried at room temperature. 4.Detection and analysis The chips were scanned with a ScanArray 4000 (GSI Lumonics, Bellerica, MA, USA) at two wavelengths, 635 and 532 nm, to detect emission from both Cy5 and Cy3, respectively. The acquired images were analyzed using GenePix Pro 3.0 software. The intensities of each spot at the two wavelengths represent the quantity of Cy3-dUTP and Cy5-dUTP. Ratios of Cy5 to Cy3 were computed using the GenePix Pro 3.0 median of ratio method. Overall intensities were normalized using the corresponding GenePix default normalization factor. All spots flagged “Bad” or “Not Found” by GenePix software were removed from the final data. Only genes with raw intensity values for both Cy3 and Cy5 of >200 were chosen for differential analysis. Genes were identified as differentially expressed if the ratio was >2 or <0.5, or the absolute value of base 2 logarithm of the ratio was >1 or < -1. mature proglottids, immature proglottids, gravid proglottids vs common reference scolex-neck proglottids tissue. 2 biological replicates for each experiments.

本研究采用互补DNA微阵列（complementary DNA microarray），分析扩展莫尼茨绦虫（Moniezia expansa）不同节片的基因表达谱。本研究共分析了4056条序列，涵盖代表新基因、已知基因及对照基因的全长与部分互补DNA（cDNA）。本研究利用cDNA微阵列检测扩展莫尼茨绦虫不同节片的基因表达谱。本研究所得的有趣数据可为更好地理解繁殖机制提供参考，并可为开发更有效的蠕虫病治疗方案提供潜在的靶标分子。

1. 微阵列构建
通过对cDNA文库中的2642条序列进行聚类分析，我们获得了1078个扩展莫尼茨绦虫单基因（unigene），涵盖代表新基因或已知基因的全长与部分cDNA，用于微阵列构建。芯片中非绦虫来源的阴性对照点为水稻基因（共48个点）。此外，芯片包含本实验室提供的9个已知基因（共54个点）作为阳性对照基因，以及24个空白点。所有单基因与对照基因均被克隆至质粒载体。本研究的DZH(TC)芯片由中国联合基因控股有限公司（中国上海）按照Li等报道的方法制备。通过使用针对质粒载体序列的通用引物进行聚合酶链式反应（PCR）扩增cDNA插入片段，随后进行纯化。所有PCR产物均通过琼脂糖凝胶电泳检测，以确保扩增克隆的质量与预期一致性。随后将扩增得到的PCR产物溶解于含3×SSC的缓冲液中。使用搭载ChipMaker微点样技术（TeleChem International，美国加利福尼亚州森尼韦尔）的Cartesian PixSys 7500运动控制点样仪（Cartesian Technologies，美国加利福尼亚州欧文），将含PCR产物的溶液点样至DZH-TC型号的载玻片上。随后将载玻片置于湿度70%的环境中水合2小时，室温晾干0.5小时，再进行紫外交联（65 mJ/cm²）。后续在室温下对载玻片进行如下处理：于0.2%十二烷基硫酸钠（SDS）溶液中浸泡10分钟，蒸馏水浸泡10分钟，0.2%硼氢化钠（NaBH₄）溶液中浸泡10分钟。再次晾干后，芯片即可用于杂交实验。

2. RNA制备与探针标记
从6条具有头节-颈节片、未成熟节片、成熟节片及孕卵节片的扩展莫尼茨绦虫中提取总RNA。将组织样品置于10 cm无RNase陶瓷研钵中研磨为细粉，随后在TRIzol试剂（Biostar，中国上海）中进行匀浆。离心后，将上清液与有机相分离，并用等体积氯仿进行萃取。取水相，加入等体积异丙醇于4℃下沉淀RNA，离心收集RNA沉淀，并用Milli-Q级水溶解。通过逆转录反应制备荧光标记的cDNA探针，使用Cy3-或Cy5-脱氧尿嘧啶三磷酸（Cy3-/Cy5-dUTP，Amersham Pharmacia Biotech，美国新泽西州皮斯卡塔韦），具体步骤如下：向体系中加入5 μg寡聚dT₁₈，将混合物加热至70℃保温10分钟使引物与3 μg mRNA退火，随后置于冰上冷却。最终反应缓冲液体系包含dNTPs（200 μM dATP、dCTP和dGTP；60 μM dTTP；以及60 μM Cy3-或Cy5-dUTP）、2 μL Superscript II逆转录酶（Invitrogen，美国加利福尼亚州卡尔斯巴德）及1×反应缓冲液（总体积10 μL）。反应于42℃下进行2小时。通过加入4 μL 2.5 M NaOH水解RNA，将混合物于65℃保温10分钟，随后用4 μL 2.5 M HCl中和。将头节-颈节片的RNA样品用Cy3-dUTP标记，未成熟节片、成熟节片及孕卵节片的RNA样品分别用Cy5-dUTP标记。将两种颜色的探针混合后用TE缓冲液稀释至500 μL，使用Microcon YM-30超滤管（Millipore，美国马萨诸塞州贝德福德）浓缩至10 μL，随后真空干燥。

3. 杂交
将探针溶解于20 mL杂交液（5×SSC，即0.75 M氯化钠与0.075 M柠檬酸钠，0.4% SDS，50%甲酰胺）中。将微阵列芯片用含0.5 mg/mL变性鲑鱼精DNA的杂交液于42℃下预杂交6小时。将荧光探针混合物于95℃变性5分钟，随后置于预杂交后的芯片上，加盖玻片。芯片于42℃下杂交15~17小时。随后将杂交后的芯片分别于60℃下用2×SSC与0.2% SDS溶液、0.1×SSC与0.2% SDS溶液、0.1×SSC溶液各洗涤10分钟，室温晾干。

4. 检测与分析
使用ScanArray 4000扫描仪（GSI Lumonics，美国马萨诸塞州贝勒里卡）分别于635 nm与532 nm两个波长下扫描，以检测Cy5与Cy3的荧光信号。使用GenePix Pro 3.0软件对获取的图像进行分析。两个波长下每个点的信号强度分别代表Cy3-dUTP与Cy5-dUTP的含量。使用GenePix Pro 3.0的比值中位数法计算Cy5与Cy3的信号比值。使用GenePix默认的标准化因子对总信号强度进行标准化。所有被GenePix软件标记为"Bad"或"Not Found"的点均被从最终数据中剔除。仅选取Cy3与Cy5的原始信号强度均大于200的基因进行差异表达分析。当信号比值大于2或小于0.5，即比值的以2为底的对数绝对值大于1或小于-1时，将该基因鉴定为差异表达基因。比较组为未成熟节片、成熟节片、孕卵节片与共同参照的头节-颈节片组织。每个实验设置2次生物学重复。