Analysis of multiple heterogeneous mRNAs in single cells.
We describe a two-stage amplification procedure designed to analyze multiple heterogeneous mRNA from single cells. Using an oligo(dT) primer with an attached phage promoter, the whole mRNA pool from a single cell was first transcriptionally amplified. This step brings the weak signal from one cell to a range in which it can be reliably picked up by the polymerase chain reaction (PCR) procedure. The cDNA was then divided for separate PCR amplification to obtain an unambiguous signal for each gene product. The linear amplification of phage transcription increased the convenience and reliability of detecting multiple messengers in the second stage. The procedure is extremely sensitive because it combines the amplification generated by both phage transcription and PCR. Using a pAW109 artificial RNA, we demonstrated that this procedure detects 10 copies of pAW109 RNA per original sample with 90% confidence and 50 copies per sample with > 95% confidence. This procedure of multiple mRNA analysis allows "phenotyping" of any cell for its mRNA composition. Examples involving several immediate early genes and subunits of the gamma-aminobutyric acidA receptor genes are given. The method should greatly facilitate the analysis of combinatorial expression of various regulatory or channel molecules in their native environments. The procedure should also provide a direct and efficient way of decoding the developmental instruction coded through combinatorial transcriptional regulation.
Laboratory of Developmental Neurobiology, NICHD, NIH, Bethesda, Maryland 20892.
H Z
HZ
Sheng
P X
PX
Lin
P G
PG
Nelson
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D005727:Ganglia, Spinal / Q000737:chemistry; D005784:Gene Amplification; D017781:Genes, Immediate-Early; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D011963:Receptors, GABA-A / Q000235:genetics; D015203:Reproducibility of Results; D013116:Spinal Cord / Q000737:chemistry
D017931:DNA Primers; D012333:RNA, Messenger; D011963:Receptors, GABA-A