A Step Ahead

Reverse transcriptase-PCR (RT-PCR) combines cDNA synthesis with amplification of target sequences as a tool for detecting mRNA expression. The sensitivity of this technique makes it especially useful for detection of low-abundance transcripts. However, RT-PCR reactions can be tedious to set up and perform. Typically, cDNAs are produced in one reaction and transferred to a second tube for PCR amplification. This procedure is time consuming and introduces multiple opportunities for cross-contamina

Jeanne Mcadara
Apr 2, 2000

Reverse transcriptase-PCR (RT-PCR) combines cDNA synthesis with amplification of target sequences as a tool for detecting mRNA expression. The sensitivity of this technique makes it especially useful for detection of low-abundance transcripts. However, RT-PCR reactions can be tedious to set up and perform. Typically, cDNAs are produced in one reaction and transferred to a second tube for PCR amplification. This procedure is time consuming and introduces multiple opportunities for cross-contamination. Even a single molecule of contaminating plasmid can turn the sensitivity of the technique into a liability.

The Advantage One-Step RT-PCR Kit from CLONTECH, of Palo Alto, Calif., was designed to get around these problems and at the same time improve the specificity and sensitivity of RT-PCR. In this system, RT and PCR steps are carried out in a single tube, so once the reaction begins, there is no need to open the tube until the experiment is complete.

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