Validation of a novel ultra-short immunolabeling method for high-quality mRNA preservation in laser microdissection and real-time reverse transcriptase-polymerase chain reaction

Abstract

Laser microdissection allows isolation of tiny samples from tissue sections for analysis of gene expression by real-time quantitative polymerase chain reaction (PCR). Although immunohistochemical labeling is often required to identify target structures, it drastically degrades mRNA so that shortened protocols are needed. Here, we present a novel method that allows fluorescence double labeling to be performed in only one incubation of 5 minutes. Fab fragments directly coupled to fluorochromes are linked to primary antibodies before these complexes are applied to sections. We quantified the influences of fixatives, labeling solutions, and incubation time on the mRNA yield and compared our method with previously proposed protocols. While tissue components, ie, vimentin and Ki67 antigen, were sufficiently stained after only 5 minutes of incubation, the new method produced a minute loss of mRNA that did not significantly differ from that of untreated sections. In contrast, incubation times of 15 and 30 minutes reduced the mRNA yield by 99.8 to 99.9%. Furthermore, incubation periods longer than 5 minutes critically affected the ratio between the target and housekeeping genes tested by factors of up to 10.6. In conclusion, the novel method described here reduces mRNA loss and potential ratio shifts to a level that does not significantly differ from that of unlabeled samples.

Figure 1

a–c: Fluorescence double labeling of vimentin (green) and the proliferation marker Ki67 (red) in rabbit appendix using the Fab fragment technique. The section was incubated for 5 minutes with a mixture of an anti-vimentin antibody coupled to Alexa Fluor 488-labeled anti-mouse Fab fragments and an anti-Ki67 antibody coupled to Alexa Fluor 555-labeled anti-mouse Fab fragments. a and b show single channel fluorescences; the overlay of the two channels is depicted in c. The presence of vimentin is used to identify the membranous (M) cells (arrows) in the dome epithelium (de). In addition, fibroblasts and some other cells of the lamina propria (lp) contain vimentin and are thus labeled, whereas the opposed non-dome epithelium (nde) is devoid of vimentin-containing cells. The nuclei of proliferating cells (red, arrowheads), most of which are lymphocytes, are located in the cytoplasmic pockets of the M cells and in the lamina propria. Imaging was done in a confocal laser scanning microscope to depict the full quality of the labeling independent of optical limitations. d and e: Fluorescence view of rabbit appendix before (d) and after (e) laser microdissection of an epithelial region (asterisk) containing M cells. The section was labeled for vimentin in a single incubation of only 5 minutes. The quality of the micrographs is limited because they were taken in a low quality video camera of the microdissection system, and because optical imaging was done through the supporting membrane, which scatters light and produces autofluorescence. The latter is additionally induced by the laser beam at the margins of the microdissected area. a–c, 250:1; d and e, 70:1.

Figure 2

Effect of different fixatives on mRNA recovery of the housekeeping gene EF-1a. Cryo-sections of rabbit appendix were incubated with a Cy3-conjugated anti-vimentin antibody for 5 minutes (one-step direct method). Each bar represents the mean Ct value and the SD of six identically treated samples. Although the different fixatives influenced the mRNA preservation only marginally, ethanol gave significantly better mRNA yields than Carnoy’s medium (P < 0.05).

Figure 3

Effect of the incubation period on EF-1a mRNA recovery. Cryo-sections of rabbit appendix were fixed in ethanol for 2 minutes and incubated with antibody solution for different periods. Prolongation of the incubation period from 5 to 60 minutes leads to a drastic loss of detectable mRNA copies, corresponding to significantly increased Ct values in real-time PCR (P < 0.05; asterisks). The difference between the 5- and 15-minute samples is 9.0 Ct units, indicating that within only 10 minutes of additional incubation, there is a loss of 99.8% of specific mRNA. Bars represent means and SD of six samples per group.

Figure 4

Impact of different staining and labeling protocols on the amount of EF-1a mRNA detected in cryo-sections of rabbit appendix using quantitative real-time RT-PCR. All methods that comprise a single incubation of only 5 minutes (bars 2 to 5) yielded comparable mRNA recovery and did not significantly differ from untreated controls. In contrast, an incubation of 60 minutes led to a significant loss of detectable EF-1a copies (P < 0.05; asterisk). The difference of 9.3 Ct units, which equals a factor of more than 600, between the 5-minute and the 60-minute incubations corresponds to a quantitative loss of 99.8% EF-1a mRNA. Bars represent means and SD of six samples per group.

Figure 5

Influence of different staining and labeling techniques on ratios between the target gene vimentin and the housekeeping gene EF-1a. Although samples that underwent labeling or staining techniques that require just 5 minutes of incubation show ratios almost identical to those obtained from untreated tissues, we found a significant shift of ratios in tissue sections that were incubated for 15, 30, or 60 minutes compared with untreated tissue (P < 0.05; asterisks). Each bar represents ratios and SD of six identically treated samples.