The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed, paraffin-embedded sections.
Laser-assisted microdissection (LAM) is now widely used to obtain specific cell populations from heterogeneous tissues. A major disadvantage of LAM is poor tissue morphology during microscopy, in part because coverslips are not used. Immunohistochemical labeling can improve identification of target cells but may affect the subsequent analysis of the microdissected tissue. We studied the effect of immunohistochemistry (IHC) on mRNA recovery from labeled cells after microdissection from both frozen and formalin-fixed, paraffin-embedded (FFPE) sections, using Melan-A and Ki-67 staining in lymph nodes with metastatic melanoma as a model. We developed rapid protocols for immunostaining in an attempt to limit loss of mRNA during procedures. A sensitive real-time quantitative reverse transcription-PCR was used to measure mRNA. We found a marked decrease in the mRNA yield from 500 microdissected cells from frozen and paraffin sections after immunostaining for both markers. Recovery of mRNA decreased by up to 89%, comparing the immunostained with the routinely stained sections. Interestingly, the ratio between mRNA for the two markers was similar in all stains, indicating that immunostained sections may be used for mRNA analysis. We also investigated the effect of storing membrane-mounted sections for microdissection under different conditions. Slides mounted with paraffin sections could be stored at room temperature for up to 90 days with no significant decrease in mRNA recovery.
Gjerdrum
Lise Mette
LM
Institute of Pathology, Aahus University Hospital, Aarhus, Denmark. lmgje@mail.dk
Abrahamsen
Helene N
HN
Villegas
Berta
B
Sorensen
Boe S
BS
Schmidt
Henrik
H
Hamilton-Dutoit
Stephen J
SJ
D000199:Actins / Q000235:genetics / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005557:Formaldehyde; D005629:Frozen Sections; D015870:Gene Expression; D016591:Histocytological Preparation Techniques / Q000379:methods*; D006801:Humans; D007834:Lasers; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000556:secondary; D042282:Microdissection / Q000379:methods*; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016707:Tissue Fixation; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
D000199:Actins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D001613:beta 2-Microglobulin; D005557:Formaldehyde