Validation of a novel ultra-short immunolabeling method for high-quality mRNA preservation in laser microdissection and real-time reverse transcriptase-polymerase chain reaction.
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.
von Smolinski
Dorthe
D
Institute of Anatomy, University of Lübeck, 23538 Lübeck, Germany.
Blessenohl
Maike
M
Neubauer
Carsten
C
Kalies
Kathrin
K
Gebert
Andreas
A
D000818:Animals; D007150:Immunohistochemistry / Q000379:methods*; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods*; D011309:Preservation, Biological; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D012333:RNA, Messenger