T7-based linear amplification of low concentration mRNA samples using beads and microfluidics for global gene expression measurements.
We have demonstrated in vitro transcription (IVT) of cDNA sequences from purified Jurkat T-cell mRNA immobilized on microfluidic packed beds down to single-cell quantities. The microfluidically amplified antisense-RNA (aRNA) was nearly identical in length and quantity compared with benchtop reactions using the same starting sample quantities. Microarrays were used to characterize the number and population of genes in each sample, allowing comparison of the microfluidic and benchtop processes. For both benchtop and microfluidic assays, we measured the expression of approximately 4000 to 9000 genes for sample amounts ranging from 20 pg to 10 ng (2 to 1000 cell equivalents), corresponding to 41 to 93% of the absolute number of genes detected from a 100 ng total RNA control sample. Concordance of genes detected between methods (benchtop vs. microfluidic) and repeats (microfluidic vs. microfluidic) typically exceeded 90%. Validation of microarray by Real-time PCR of a panel of five genes suggests transcription of genes present is approximately six times more efficient with the microfluidic IVT compared with benchtop processing. Microfluidic IVT introduces no bias to the gene expression profile of the sample and provides more efficient transcription of mRNA sequences present at the single-cell level.
Kralj
Jason G
JG
National Institute of Standards and Technology, 100 Bureau Dr MS 8313, Gaithersburg, MD, USA. jason.kralj@nist.gov
Player
Audrey
A
Sedrick
Hope
H
Munson
Matthew S
MS
Petersen
David
D
Forry
Samuel P
SP
Meltzer
Paul
P
Kawasaki
Ernest
E
Locascio
Laurie E
LE
D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000032:analysis* / Q000235:genetics; D016372:RNA, Antisense / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins
D018076:DNA, Complementary; D016372:RNA, Antisense; D012333:RNA, Messenger; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases