mRNA Amplification and Quantitation using URT-PCR
A new method has been developed to detect gene expression for human tissue in mRNA without genomic DNA contamination. This technology makes true quantitation of gene expression in human tissue of any size accurate and feasible.
Researchers have devised a novel reverse transcription strategy that utilizes a mRNA-specific RT primer designed to be universal for all mRNA transcripts. The employment of the mRNA-specific RT strategy obviates the limitations posed by total RNA preps contaminated with gDNA. Subsequent PCR is performed integrating features of the universal RT primer design, and is not complicated by the presence of gDNA nor pseudogene. Messager RNA can thus be amplified very specifically in the setting of gDNA contamination, without further steps. Purification of total RNA to mRNA becomes unnecessary, a particularly critical advantage when handling small tissue samples, as in human tissues. Both DNase treatment after RNA isolation, and no-RT controls in the PCR step to assess the presence of pseudogene, become unnecessary. Standard reference “housekeeper” transcripts such as β-actin and GAPDH, otherwise contaminated by gDNA-encoding pseudogenes, can be amplified specifically and quantitatively. Multiple transcripts can be amplified from the same total RNA aliquot, maximizing the efficiency of reverse transcription on limited RNA samples.
The universal reverse transcription strategy is quantitatively similar in efficiency to conventional oligo (dT) primer tags. PCR primer design is not further complicated by the presence of pseudogene sequences competing for PCR primer. Genomic DNA – encoded pseudogene sequences that exist for reference gene transcripts (e.g. β-actin and GAPDH) and target gene transcripts (e.g. GSTM1 and GSTP1) have been amplified alongside non-pseudogene-encoded sequences (e.g. CYP1B1, NQO1) by otherwise conventional PCR techniques, with the only change being the use of different RT and PCR primers.
The system has been readily employed in the common and currently used qualitative and real-time quantitative PCR protocols assessing gene expression in small samples of human tissues. The strategy has therefore proven to be universally applicable to all tested transcripts in all tested gene-expression assay contexts.
- Gene expression
- Genetic regulation
- Expression arrays
- Human gene expression profiling
- Quantitative RT-PCR
- Gene-environment interaction
- URT for all tested transcripts
- mRNA specific PCR
- Standard RNA isolation techniques are acceptable – no additional purification steps
- Multiple transcripts can be amplified from one total RNA sample
- Standard housekeeper reference transcripts (i.e. β-actin, GAPDH) are valid controls
- Usable with standard RT-PCR strategies/kits – only primers differ
- Quantitation of low copy transcripts in small tissue samples is feasible
- Sensitivity similar to standard RT-PCR protocols
Potential Areas of Application
- Gene expression assays, from traditional to microarrays
- Quantitative RT-PCR applications
- Gene regulation research
- Gene expression phenotyping of cells and tissues for research or clinical molecular pathology
- Small sample (e.g. micro dissection) or single cell expression assays
- Inter-individual expression phenotyping for disease susceptibility
State of Development
- Fully operational universal reverse transcription primer at research scale
- PCR primers specifically designed for >12 transcripts also at research scale
- Inter-individual (n>50) sample performance verified, and subset quantified
- Performance versus standard oligo (dT) strategy quantified
- All relevant controls demonstrated
USPTO no. 7,141,372
HRI is seeking commercial partners to develop of this technology for clinical and commercial use. Available for licensing.
Simon D. Spivack, PhD
Gregory J. Hurteau
Diane L. Borghoff, B.S., M.S.
Marketing & Licensing Associate – Intellectual Property
Health Research, Inc. – 150 Broadway – Suite 560, Menands, New York 12204-2719 U.S.A.
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