Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue.
BACKGROUND
Analysis of renal biopsies is currently based on histological recognition of typical structural patterns and immunohistological detection of protein expression alterations. Both can be performed using formaldehyde as the tissue fixative. As a consequence of recent advances in molecular medicine, mRNA expression analysis may offer an attractive option to obtain functionally relevant information. However, quantification of mRNA expression in human renal biopsies thus far has not been possible in formaldehyde-fixed tissue.


METHODS
The present study evaluated a recently reported mRNA extraction protocol. Using this approach gene expression analysis could be performed on formaldehyde-fixed archival renal tissues by laser microbeam microdissection, laser pressure catapulting and real time reverse transcription-polymerase chain reaction.


RESULTS
For an initial feasibility study, the expression of two chemokines (IP-10 and RANTES) in renal transplant rejection was examined. Induction of protein expression in allografts undergoing rejection was demonstrated for both chemokines by immunohistochemistry. The mRNA expression alterations in the defined renal compartments of glomeruli, vessels and tubulointerstitium were quantified using laser microdissection from formaldehyde-fixed, paraffin-embedded or frozen tissue sections. A pronounced increase of mRNA expression compared to controls was demonstrated for IP-10 as well as RANTES with both tissue-processing protocols.


CONCLUSIONS
Using formaldehyde as the tissue fixative, information on the disease process can now be obtained by histological, immunohistochemical and gene expression techniques. In the future this may allow the study of activated molecular programs in routine renal biopsies as well as archival tissue samples.
Cohen
Clemens D
CD
Medizinische Poliklinik, Ludwig-Maximilians-University of Munich, Munich, Germany.
Gröne
Hermann-Josef
HJ
Gröne
Elisabeth F
EF
Nelson
Peter J
PJ
Schlöndorff
Detlef
D
Kretzler
Matthias
M
D000293:Adolescent; D000328:Adult; D001109:Archives; D001706:Biopsy / Q000379:methods*; D018946:Chemokine CCL5 / Q000235:genetics; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000235:genetics; D004210:Dissection / Q000379:methods; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D015870:Gene Expression; D006084:Graft Rejection / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000473:pathology*; D007674:Kidney Diseases / Q000473:pathology; D016030:Kidney Transplantation; D007834:Lasers; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018946:Chemokine CCL5; D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D005404:Fixatives; D012333:RNA, Messenger; D005557:Formaldehyde