Expression profiling across many samples via manifold-assisted mRNA processing.
Analysis of mRNA provides a condensed view of gene structure, and quantitative analyses can reveal induction of physiological or pathological gene expression programs. One of the main hurdles for routine mRNA analyses is the need to prepare large sets of samples in a rapid and standardized manner. We describe here a procedure for mRNA isolation and cDNA synthesis using manifold devices, consisting of a set of prongs that project into individual reaction wells. The prongs have a high binding capacity for the polyA-tails of mRNA and the captured mRNA is directly used to synthesize cDNA on the supports, followed by amplification. The convenience and reproducibility of the procedure allows profiling of gene expression over time, by comparing many different samples. Using the device mRNA was simultaneously isolated and accurately measured from up to 96 different samples of anywhere between 10 and 200 000 cells. The amounts of a leukemia-specific transcript could be measured when the malignant cells represented </=0.01% of the sampled cells. We illustrate the possibility of analyzing a number of tissues and monitoring expression of sets of cytokines, involved in rejection, at variable times after transplantation.
Hagberg
A
A
Department of Genetics and Pathology and Department of Clinical Immunology, Rudbeck Laboratory, Se-75185 Uppsala, Sweden.
Barbany
G
G
Krook
H
H
Samiotaki
M
M
Landegren
U
U
D000818:Animals; D002478:Cells, Cultured; D002482:Cellulose / Q000031:analogs & derivatives*; D016207:Cytokines / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis; D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000235:genetics; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods; D006801:Humans; D016381:Islets of Langerhans Transplantation / Q000276:immunology; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D011061:Poly A; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D011917:Rats, Inbred Lew; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014183:Transplantation, Heterologous / Q000276:immunology; D014407:Tumor Cells, Cultured
D016207:Cytokines; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C021722:oligo(dT)-cellulose; D011061:Poly A; D002482:Cellulose; D016044:Fusion Proteins, bcr-abl