Analyses Of The RNA Extracted By Different Methods From Various Pathological Sections And Glomeruli By Laser Capture Microdissection And Real Time Quantitative PCR

Background and objective: Pathological sections, which are widely being used to research the kidney diseases, are more valuable when the gene expression in total sections or special structures can be measured. The RNA isolated successfully is so crucial to measure gene expression that we analyze the effect of different isolate methods and various fixation buffers on the RNA in pathological sections and glomeruli quantitatively and site-specifically by a laser capture microdissection and quantitative PCR.Methods: Kidneys, which were collected from six 250g weighted Wistar rats, were disposed to make several kinds of paraffin-embedded sections and frozen sections. For paraffin-embedded sections, which were fixed separately by three different buffers such as acetone , methanol-chloroform-glacialacetic acid (methacarn), 10% neutral-buffered formalin(NBS), RNA was isolated by RNA lysis buffer and Trizol Agent separately, and after that,180bp GAPDH gene and 565bp beta-actin gene was amplified by RT-PCR for detecting its quality, and GAPDH was measured by SYBR GREEN 1 quantitative RT-PCR for detecting its quantity. For Glomeruli isolated by laser capture microdissection (LCM) from different frozen sections, which were fixed by methanol,70% ethacol,acetone,4% paraformaldehyde, RNA was extracted by GTC and Trizol agent methods and analyzed by measuring GAPDH gene expression by Taqman quantitative PCR. For comparing the RNA of Glomeruli in between acetone-fixed paraffin sections and frozen sections, GAPDH gene was measured by Taqman quantitative PCR Results We had found that, for a whole paraffin section, 180bp GAPDH gene and 565bp beta-acting gene could successfully be amplified by RT-PCR; when using RNA lysis buffer, more RNA was obtained than Trizol Agent and there was nodifference of RNA among various buffer-fixed sections, When using Trizol Agent to isolate RNA, precipitate fixatives such as acetone-fixed sections were best, cross-linking fixatives such as NBS were worse , and methacarn was medium; for RNA of glomeruli in frozen sections, no significant difference was found among sections which were fixed by precipitate fixatives such as methanol, ethanol, and acetone when using GTC or Trizol Agent to extract RNA, however, for cross-linking fixative such as 4% formaldehyde-fixed sections , Trizol Agent was better than GTC to acquire more RNA which had no significant difference with precipitate fixatives fixed sections; when comparing RNA of glomeruli in between acetone-fixed paraffin-embedded and frozen sections, there was no significant difference.Discussion: In conclusion, we inclined to claim that it is important to select suitable RNA extract methods for different fixations to extract RNA .In this project, through isolating the RNA and analyzing the RNA in various kinds of pathological sections and glomeruli by laser microdissection and quantitative PCR, we provide some valuable information about both how to isolate the RNA and detect the gene expression in total sections or glomeruli successfully.