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

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 lysis 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 coamplify 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 ≤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).