Prospects of transcript profiling for mRNAs and MicroRNAs using formalin-fixed and paraffin-embedded dissected autoptic multiple sclerosis lesions.
The elaboration of novel pathogenic aspects of multiple sclerosis (MS) requires the analysis of well-defined stages of lesion development. However, specimens of certain stages and lesion types are either present in small brain biopsies, insufficient in size for further molecular studies or available as formalin-fixed and paraffin-embedded (FFPE) material only. Therefore, application of current molecular biology techniques to FFPE tissue is warranted. We compared FFPE and frozen tissue by using quantitative polymerase chain reaction and report: (1) FFPE material is highly heterogeneous regarding the utility for transcript profiling of mRNAs; well-preserved FFPE samples had about a 100-fold reduced sensitivity compared with frozen tissue, but gave similar results for genes of sufficient abundance; (2) FFPE samples not suitable for mRNA analysis are still highly valuable for miRNA quantification; (3) the length of tissue fixation greatly affects utility for mRNA but not for miRNA analysis; (4) FFPE samples can be processed via a hot water bath for dissection of defined lesion areas; and (5) in situ hybridization for proteolipid protein (PLP) helps to identify samples not suitable for mRNA amplification. In summary, we present a detailed protocol how to use autoptic FFPE tissue for transcript profiling in dissected tissue areas.
Institute of Clinical Neuroimmunology, University Hospital Großhadern, Ludwig-Maximilians-University, Munich, Germany.
Sylvia
S
Eisele
Markus
M
Krumbholz
Marie-Therese
MT
Fischer
Hema
H
Mohan
Andreas
A
Junker
Thomas
T
Arzberger
Reinhard
R
Hohlfeld
Monika
M
Bradl
Hans
H
Lassmann
Edgar
E
Meinl
D001344:Autopsy; D005260:Female; D005557:Formaldehyde; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000378:metabolism*; D009103:Multiple Sclerosis / Q000473:pathology*; D018991:Myelin Proteolipid Protein / Q000235:genetics / Q000378:metabolism; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods
D035683:MicroRNAs; D018991:Myelin Proteolipid Protein; C472930:PLP1 protein, human; D012333:RNA, Messenger; D005557:Formaldehyde