Use of Flow Cytometry and RT-PCR for Detecting Gene Expression by Single Cells

Abstract

We have developed a method for reliably detecting gene expression by individual, phenotypically defined cells. Cells were sorted by flow cytometry into 96-well plates containing a Nonidet P-40 (NP40)-based bysis solution. Reverse transcription (RT) of cellular major histocompatibility complex class II DQB and either bovine leukemia virus (BLV) env or tax/rex mRNA was subsequently conducted using gene-specific oligonucleotide primers. Two sequential rounds of PCR were then performed to co-amplify DQB and either BLV env or tax/rex cDNA. The PCR products were electrophoresed in 6% polyacrylamide gels and visualized by ethidium bromide staining. The BLV-infected BL3 cell line was used to establish the sensitivity of the method; cellular and viral mRNA were reproducibly detected in wells into which single BL3 cells were sorted. Additionally, BLV env mRNA from single infected cells was consistently detected in reactions containing as many as 1000 uninfected cells. By using this method, 0.012% +/- 0.002% of B cells from a BLV-infected cow with persistent lymphocytosis were found to express BLV tax/rex mRNA, whereas < or = 0.001% expressed BLV env mRNA. The combination of single-cell sorting and RT-PCR provides a powerful new tool to study viral transcription, host responses associated with progression of retroviral infections or other problems requiring determination of the frequency of cells expressing a particular gene(s).

Dissemination of Bovine Leukemia Virus-Infected Cells from a Newly Infected Sheep Lymph Node

“…3). Although BLV expression cannot be detected in most infected mononuclear cells circulating in the blood of infected sheep or cows (14,32,46), cells leaving a newly infected lymph node might actively produce virus. To explore this possibility, we immunostained lymph cells freshly isolated from an infected sheep for BLV CA protein.…”

Cell dynamics and immune response to BLV infection: a unifying model

“…First, BLV virions or viral proteins cannot be directly detected in the peripheral blood by any available technique (ELISA, flow cytometry, immunoprecipitation or Western blotting). Second, viral transcripts from peripheral blood lymphocytes or tumors can only be amplified by means of very sensitive RT-PCR techniques (15,57,58). Third, using flow cytometry cell sorting and subsequent RT-PCR, only about one B lymphocyte out of 10,000 is found to express tax/rex mRNA during persistent lymphocytosis (57).…”

“…Second, viral transcripts from peripheral blood lymphocytes or tumors can only be amplified by means of very sensitive RT-PCR techniques (15,57,58). Third, using flow cytometry cell sorting and subsequent RT-PCR, only about one B lymphocyte out of 10,000 is found to express tax/rex mRNA during persistent lymphocytosis (57). Fourth, only rare cells in the peripheral blood (1 in 50,000) contain enough BLV transcripts to be identified readily by in situ hybridization (59,60).…”

Applications of Flow Cytometry to Clinical Microbiology

“…FCM and RT-PCR have detected gene expression in individual cells (111). Muratori et al (225) developed an in situ RT-PCR technique using fluorescein-labeled HCV specific primers detected by FCM for the quantification and phenotyping of HCV-infected cells in clinical blood samples.…”