Use of flow cytometry and RT-PCR for detecting gene expression by single cells.

Important note: Information in this article was accurate in 1997. The state of the art may have changed since the publication date.

Use of flow cytometry and RT-PCR for detecting gene expression by single cells.

Biotechniques. 1996 Aug;21(2):286-91. Unique Identifier : AIDSLINE MED/97016177
Gaynor EM; Mirsky ML; Lewin HA; University of Illinois at Urbana-Champaign, USA.

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).

Keywords: *Cell Separation/METHODS *Cells/METABOLISM *Flow Cytometry/METHODS *Gene Expression *Polymerase Chain Reaction/METHODS *RNA, Messenger/BIOSYNTHESIS
970630
M9761242

AEGiS is a 501(c)3, not-for-profit, tax-exempt, educational corporation. AEGiS is made possible through unrestricted funding from Boehringer Ingelheim, Bridgestone/Firestone Charitable Trust, Bristol-Myers Squibb Company, Elton John AIDS Foundation, Gill Foundation, the National Library of Medicine, Quest Diagnostics, Roche and Trimeris, and donations from users like you. Always watch for outdated information. This article first appeared in 1997. This material is designed to support, not replace, the relationship that exists between you and your doctor.

AEGiS presents published material, reprinted with permission and neither endorses nor opposes any material. All information contained on this website, including information relating to health conditions, products, and treatments, is for informational purposes only. It is often presented in summary or aggregate form. It is not meant to be a substitute for the advice provided by your own physician or other medical professionals. Always discuss treatment options with a doctor who specializes in treating HIV.

Copyright ©1980, 1997. AEGiS. All materials appearing on AEGiS are protected by copyright as a collective work or compilation under U.S. copyright and other laws and are the property of AEGiS, or the party credited as the provider of the content. .