کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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5522047 | 1545667 | 2017 | 5 صفحه PDF | دانلود رایگان |
- A method for obtaining lentiviral RNA from LCM-captured laser capture micro dissected CD68Â + and CD163Â + macrophages
- A modified method using rapid immunohistochemistry in the presence of RNase-inhibitors
Laser capture microdissection (LCM) is used to extract cells or tissue regions for analysis of RNA, DNA or protein. Several methods of LCM are established for different applications, but a protocol for consistently obtaining lentiviral RNA from LCM captured immune cell populations is not described. Obtaining optimal viral RNA for analysis of viral genes from immune-captured cells using immunohistochemistry (IHC) and LCM is challenging. IHC protocols have long antibody incubation times that increase risk of RNA degradation. But, immune capture of specific cell populations like macrophages without staining for virus cannot result in obtaining only a fraction of cells which are productively lentivirally infected. In this study we sought to obtain simian immunodeficiency virus (SIV) RNA from SIV gp120Â + and CD68Â + monocyte/macrophages in bone marrow (BM) and CD163Â + perivascular macrophages in brain of SIV-infected rhesus macaques. Here, we report an IHC protocol with RNase inhibitors that consistently results in optimal quantity and yield of lentiviral RNA from LCM-captured immune cells.
Journal: Journal of Immunological Methods - Volume 442, March 2017, Pages 59-63