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 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).
Gaynor
E M
EM
University of Illinois at Urbana-Champaign, USA.
Mirsky
M L
ML
Lewin
H A
HA
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D002477:Cells / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D005802:Genes, MHC Class II; D015752:Genes, env; D016355:Genes, pX; D001909:Leukemia Virus, Bovine / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012367:RNA, Viral