Biomaterial Database

Competitive reverse-transcriptase polymerase chain reaction without an artificial internal standard. 1995

M E Zenilman, and W Graham, and K Tanner, and A R Shuldiner

Department of Surgery, Johns Hopkins University, Hopkins Bayview Medical Center, Baltimore, Maryland 21224.

Advances in our understanding of molecular and cellular physiology necessitate that mRNA levels for specific growth factors and other rare transcripts be measured quantitatively in small samples. Conventional methods such as Northern blot analysis and solution hybridization/ribonuclease protection are not sufficiently sensitive. We now report the theory, development, and validation of a rapid and highly sensitive assay, the RNA/DNA quantitative polymerase chain reaction (RD-PCR), which uses a competitive PCR approach to measure the number of copies of a specific mRNA per cell. Total nucleic acid (RNA and genomic DNA) is isolated from cells in culture. The mRNA of interest is first reverse-transcribed with an oligomer bearing a complementary sequence specific for the mRNA at its 3'-end, and a sequence complementary to an intron of the desired gene at the 5'-end. Competitive PCR is then performed in the presence of the cDNA product and endogenous genomic DNA, with an upstream primer complementary to the exon sequence of the gene of interest, and a downstream primer complementary to the intron sequence that was tagged to the cDNA. The cell's own genomic DNA is thereby used as the internal standard. To control for the efficiency of reverse transcription, a standard curve is used in each assay. The technique was validated by comparing the quantitation of insulin-like growth factor I (IGF-I) mRNA in two human cell lines by RD-PCR and by RNase protection analysis. Both methods gave similar numbers of copies of IGF-I mRNA per cell. For accurate analysis, RNase protection required at least 10(7) cells; RD-PCR required as little as 10(2) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012313	RNA	A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed)	RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D014158	Transcription, Genetic	The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.	Genetic Transcription
D016133	Polymerase Chain Reaction	In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.	Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain