| Description |
The histologic picture of glomerulonephritis is characterized by pathological accumulation of extracellular matrix with damaged glomeruli. Previous studies have demonstrated that increased production of mesangial matrix in rat acute glomerulonephritis is caused by increased expression of transforming growth factor beta one (TGHbeta1) messenger RNA. The most popular method currently used in measuring mRNA levels is Northern blot. An abundant RNA requirement (15-30 ug), however, restricts the application of Northern blot in which amounts of isolated RNA or tiny, such as in studies using human biopsies. In this thesis project, a competitive PCR method has been developed to overcome the disadvantage of Northern blot. The internal standards, PCR mimics, or TGFbeta1, extra domain A containing fibronectin (EDA fibronectin), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were synthesized using a PCR minmic™ construction kit from CLONTECH Laboratories, Inc. They were applied as competitors of target cDNAs in measurement of the corresponding mRNA levels to correct the variations of PCR efficiency. The variations of the efficiency of reverse transcription and the variations of the quantity and quality of mRNA samples were corrected by measuring the relative levels of gene expression using the ratios of target cDNA to G3PDH cDNA. The specificity of this method was confirmed by DNA size measurement and Southern blot. The detectable amount of specific mRNA was 10[-18) moles. The amount of total mRNA applied was less than 100ng. The accuracy verified by measuring the concentrations of DNA standards was 81%. The precision confirmed by multiple measurements was 85%/ In rat acute glomerulonephritis, it was found that relative mRNA levels of TGFbeta1 started increasing on day 3(3.2-fold of normal), reached peak on day 7 (4.5-fold), and returned toward normal on day 14 (2.3-fold) and on day 21 (1.5-fold) post-ATS injection. The relative levels of EDA fibronectin, however, remained normal on day 3 (1.2-fold of normal), increased profoundly on day 7 (79.9-fold), and returned toward normal on day 14 (21.9-fold) and on day 21 (6.7-fold) post-ATS injection. The increase in TGFbeta1 and EDA fibronectin mRNA levels are significant except that in TGFbeta1 mRNA on day 21 and EDA fibronectin mRNA on day 3 and day 21. The results were comparable with Northern blot data and further supported the previous conclusion the TGFbeta1 stimulated the expression of EDA fibronection in rat acute glomerulonephritis. |
