Quantification of MUC2 and MUC5AC transcripts in human conjunctiva

PURPOSE. Transcripts of mucins 1, 4, and 5AC have been identified in human conjunctival tissue. Of these, only MUC5AC has been localized to goblet cel ls. MUC2 is a goblet cell mucin originally identified in the intestinal muc osa. The presence of MUC2 transcripts and levels of MUC2 and MUC5AC transcr ipts 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 year s, all white) was isolated and subjected to competitive reverse transcripti on-PCR. Internal standards, which are dsDNA molecules with ends complementa ry to a given primer pair but containing nonhomologous central sequences, w ere 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, beta(2)-microglobulin (beta(2)M). The identity of PCR p roducts was confirmed by sequencing. RESULTS. In the six individual samples tested, beta(2)M mRNA is expressed, on average, at approximately 10(-20) moles per sample (1 mu g RNA) or appro ximately 63.5 x 10(4) molecules. The mRNA encoding MUC5AC, a relatively abu ndant ocular mucin, exists at levels 10-fold lower than beta(2)M. In contra st to previous reports of MUC2 mRNA being absent at the ocular surface, the se results show that MUC2 transcripts are present and expressed at levels 5 900-fold lower than for MUC5AC. Apparently, MUC2 transcripts exist on the o rder of only approximately 100 to 1000 molecules/mu g of RNA in the analyze d samples. CONCLUSIONS. MUC2 transcripts are present in human conjunctival tissue and their abundance is much lower than that of MUC5AC. This is the first applic ation of competitive PCR to the quantitative analysis of mucin expression i n 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 comparis on of relative levels of expression for ocular mucins.