Abstract
PURPOSE: Lower urinary tract symptoms (LUTS) can be caused by structural and functional changes in different compartments of the bladder. To enable extensive investigations of individual regions even in small bladder biopsies, we established a combination protocol consisting of three molecular techniques: laser capture microdissection microscopy (LCM), RNA preamplification and quantitative polymerase chain reaction (qPCR).
METHODS: Urinary bladders of ten mice were resected and frozen immediately or after a delay of 15 min. Cryosections were obtained and smooth muscle was isolated using the LCM technique. Then, RNA was extracted, including protocols with and without DNase digestion as well as with and without the addition of carrier RNA. Extracted RNA was either used for reverse transcriptase (RT)-PCR plus qPCR or for a combination of RNA preamplification and qPCR.
RESULTS: Our data showed that with RNA preamplification, 10 μg cDNA can be regularly generated from 2.5 ng RNA. Depending on expression levels, this is sufficient for hundreds of pPCR reactions. The efficiency of preamplification, however, was gene-dependent. DNase digestion before preamplification lead to lower threshold cycles in qPCR. The use of partly degraded RNA for RNA preamplification did not change the results of the following qPCR.
CONCLUSIONS: RNA preamplification strongly enlarges the spectrum of genes to be analyzed in distinct bladder compartments by qPCR. It is an easy and reliable method that can be realized with standard laboratory equipment. Our protocol may lead in near future to a better understanding of the pathomechanisms in LUTS.
Horstmann, M