Reverse Transcription and PCR


The ability to study comparatively the expression of messenger RNA (mRNA) populations from different samples has become accessible by the novel application of reverse transcription (RT), together with the polymerase chain reaction (PCR), to isolate gene sequences which can then be visualized by displaying on a sequencing gel. Differentially expressed transcripts can be analysed by Northern blotting or semiquantitative PCR to confirm differential expression. Utilization of various primer combinations facilitates the isolation of approximately 15 000 mRNA species, which represents gene expression in a mammalian cell at any one time.

Keywords: mRNA

Figure 1.

An overnight exposure of a differential display polyacrylamide gel. RT‐PCR with an oligo dT‐primer and a random primer utilizing α‐32P‐labelled dATP. The gel was run for 2 h at 1200 V; each lane represents one reaction.

Figure 2.

The protocol for differential display.



Agarwal SK, Cogburn LA and Burnside J (1995) Comparison of gene expression in normal and growth hormone receptor‐deficient dwarf chickens reveals a novel growth hormone regulated gene. Biochemical and Biophysical Research Communications 206: 153.

Aliello LP, Robinson GS, Lin Y, Nisho Y and King GL (1994) Identification of multiple genes in bovine retinal pericytes altered by exposure levels of glucose by using mRNA differential display. Proceedings of the National Academy of Sciences of the USA 91: 6231.

Burn TC, Petrovick MS, Hohaus S, Rollins BJ and Tenet DG (1994) Monocyte chemoattractant protein‐1 gene is expressed in activated neutrophils and retinoic acid‐induced human myeloid cell lines. Blood 84: 2776.

Chaqour HS and Macarak EJ (1999) Identification of stretch‐responsive genes in pulmonary smooth muscle cells by a two arbitrary primer‐based mRNA differential display approach. Molecular and Cellular Biochemistry 197: 87–96.

Hagen H, Klager S, McKerrow J and Ham P (1997) Simulium damosum s.l.: isolation and identification of prophenoloxidase following an infection with Onchocerca spp. using targeted differential display. Experimental Parasitology 86: 213.

Kornmann B, Preitner N, Rifat D, Fleury‐Olela F and Schibler U (2001) Analysis of circadian liver gene expression by ADDER, a highly sensitive method for the display of differentially expressed mRNAs. Nucleic Acids Research 29: E51‐1.

Liang P and Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967.

Liang P and Pardee AB (1995) Recent advances in differential display. Current Biology 7: 274.

Liang P, Averboukh L and Pardee AB (1993) Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Research 21: 3269.

Liang P, Zhu W, Zhang X et al. (1994) Differential display using one‐base anchored oligo‐dT primers. Nucleic Acids Research 22: 5763.

Malhotra K, Fotz L, Malhoney W and Schueler P (1998) Interaction and effect of annealing temperature on primers used in differential display RT‐PCR. Nucleic Acids Research 26: 854.

Rohrwild M, Alpan RS, Liang P and Pardee AB (1995) Inosine containing primers for mRNA differential display. Trends in Genetics 11: 8.

Trentmann SM, van der Knapp E and Kende H (1995) Alternatives to 35S as a label for the differential display of eukaryotic messenger RNA. Science 267: 1186.

Vila‐Ortiz GJ, Radrizzani M, Carminatti H, Idoyaga‐Vargas VP and Santa‐Coloma TA (2001) Single strand mRNA differential display (SSDD) applied to the identification of serine/threonine phosphatases regulated during cerebellar development. Journal of Neuroscience Methods 105: 87.

Villeponteau B, Junli F, Funk W and Linskens MHK (1997) Method and kit for enhanced differential display. Biotechnology Advances 15: 476.

von der Krammer H, Albrecht C, Mayhaus M et al. (1999) Identification of genes regulated by muscarinic acetylcholine receptors: application of an improved and statistically comprehensive mRNA differential display technique. Nucleic Acids Research 27: 2211–2218.

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How to Cite close
Woodgate, L, and Mills, K(Mar 2003) Reverse Transcription and PCR. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0003751]