Gjerdrum Lise Mette, Abrahamsen Helene N, Villegas Berta, Sorensen Boe S, Schmidt Henrik, Hamilton-Dutoit Stephen J
Institute of Pathology, Aahus University Hospital, Aarhus, Denmark.
Diagn Mol Pathol. 2004 Dec;13(4):224-33. doi: 10.1097/01.pdm.0000134779.45353.d6.
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.
激光辅助显微切割（LAM）现已广泛用于从异质性组织中获取特定细胞群体。LAM的一个主要缺点是显微镜检查期间组织形态不佳，部分原因是未使用盖玻片。免疫组织化学标记可以改善靶细胞的识别，但可能会影响显微切割后组织的后续分析。我们以转移性黑色素瘤淋巴结中的Melan-A和Ki-67染色为模型，研究了免疫组织化学（IHC）对冷冻及福尔马林固定石蜡包埋（FFPE）切片显微切割后标记细胞mRNA回收的影响。我们开发了快速免疫染色方案，试图在操作过程中限制mRNA的损失。使用灵敏的实时定量逆转录PCR来测量mRNA。我们发现，对两种标记物进行免疫染色后，冷冻切片和石蜡切片中500个显微切割细胞的mRNA产量显著下降。与常规染色切片相比，免疫染色切片的mRNA回收率降低了89%。有趣的是，所有染色中两种标记物的mRNA比例相似，这表明免疫染色切片可用于mRNA分析。我们还研究了在不同条件下储存用于显微切割的膜载切片的影响。载有石蜡切片的玻片可在室温下保存长达90天，mRNA回收率无显著下降。
