Quantification of MUC2 and MUC5AC transcripts in human conjunctiva.
Quantification of MUC2 and MUC5AC transcripts in human conjunctiva.
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DOI：
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发表时间：
2000-03
影响因子：
4.4
通讯作者：
R. W. McKenzie;J. Jumblatt;M. Jumblatt
中科院分区：
文献类型：
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作者：
R. W. McKenzie;J. Jumblatt;M. Jumblatt
目的在人结膜组织中鉴定粘蛋白1、4和5AC的转录本。其中，只有MUC 5AC定位于杯状细胞。MUC 2是最初在肠粘膜中鉴定的杯状细胞粘蛋白。本文报道了用定量聚合酶链反应（PCR）测定的正常人结膜中MUC 2转录本和MUC 2及MUC 5AC转录本的水平。方法从6例（男3例，女3例，年龄44 ~ 69岁，均为白色）供者的结膜组织中提取RNA，进行竞争性逆转录PCR。为每个测定的基因制备内标物，其是末端与给定引物对互补但含有非同源中心序列的dsDNA分子。针对内标物的连续稀释液滴定恒定量的cDNA允许定量模板cDNA。将MUC 2和MUC 5AC水平与“管家”基因β 2-微球蛋白（β 2 M）的水平进行比较。通过测序确认PCR产物的身份。结果在测试的六个单独样品中，平均每个样品（1微克RNA）表达约10（-20）摩尔或约63.5x10（4）分子的β 2 M mRNA。编码MUC 5AC（一种相对丰富的眼粘蛋白）的mRNA以比β 2 M低10倍的水平存在。与先前报道的MUC 2 mRNA在眼表面不存在相反，这些结果表明MUC 2转录物存在并且以比MUC 5AC低5900倍的水平表达。显然，在分析的样品中，MUC 2转录物仅以大约100至1000个分子/微克RNA的数量级存在。结论人结膜组织中存在MUC 2转录本，其丰度远低于MUC 5AC。这是竞争性PCR首次应用于人眼组织粘蛋白表达的定量分析。该方法的灵敏度允许检测MUC 2转录本，而这些转录本在以前的研究中不能通过北方印迹分析或原位杂交检测到。它还使得比较眼粘蛋白的相对表达水平成为可能。
PURPOSE Transcripts of mucins 1, 4, and 5AC have been identified in human conjunctival tissue. Of these, only MUC5AC has been localized to goblet cells. MUC2 is a goblet cell mucin originally identified in the intestinal mucosa. The presence of MUC2 transcripts and levels of MUC2 and MUC5AC transcripts in normal human conjunctiva, as determined by quantitative polymerase chain reaction (PCR), is reported. METHODS RNA from conjunctivae of six donors (3 men, 3 women, 44 to 69 years, all white) was isolated and subjected to competitive reverse transcription-PCR. Internal standards, which are dsDNA molecules with ends complementary to a given primer pair but containing nonhomologous central sequences, were prepared for each gene assayed. Titration of a constant amount of cDNA against serial dilutions of the internal standard allowed quantification of the template cDNA. MUC2 and MUC5AC levels were compared to levels of the "housekeeping" gene, beta2-microglobulin (beta2M). The identity of PCR products was confirmed by sequencing. RESULTS In the six individual samples tested, beta2M mRNA is expressed, on average, at approximately 10(-20) moles per sample (1 microg RNA) or approximately 63.5x10(4) molecules. The mRNA encoding MUC5AC, a relatively abundant ocular mucin, exists at levels 10-fold lower than beta2M. In contrast to previous reports of MUC2 mRNA being absent at the ocular surface, these results show that MUC2 transcripts are present and expressed at levels 5900-fold lower than for MUC5AC. Apparently, MUC2 transcripts exist on the order of only approximately 100 to 1000 molecules/microg of RNA in the analyzed samples. CONCLUSIONS MUC2 transcripts are present in human conjunctival tissue and their abundance is much lower than that of MUC5AC. This is the first application of competitive PCR to the quantitative analysis of mucin expression in human ocular tissue. The sensitivity of this method allows the detection of MUC2 transcripts that were not detected by Northern blot analysis or in situ hybridization in previous studies. It also makes possible the comparison of relative levels of expression for ocular mucins.