Technical noteA method for obtaining simian immunodeficiency virus RNA sequences from laser capture microdissected and immune captured CD68Â + and CD163Â + macrophages from frozen tissue sections of bone marrow and brain

- A method for obtaining lentiviral RNA from LCM-captured laser capture micro dissected CD68 + and CD163 + 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 gp120 + and CD68 + monocyte/macrophages in bone marrow (BM) and CD163 + 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.