() First strand cDNA is initiated by priming with an oligo dT primer containing and anchoring primer site

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling"</p><p>Nucleic Acids Research 2006;34(3):996-1014.</p><p>Published online 9 Feb 2006</p><p>PMCID:PMC1363777.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The template switch effect is applied to incorporate a primer containing both an anchoring primer site and a RNA polymerase binding site. The anchored priming sites are used in a limited PCR cycling step. Sense RNA (sRNA) is transcribed by SP6-RNA polymerase during an reaction. Adapted figure from Rajeevan . (). () The first round of this procedure is equivalent to the first round of the classical Eberwine procedure and RNA in the antisense direction is synthesized in the reaction. At the start of the second round of amplification, aRNA is primed with random nonamer primers modified by the addition of an upstream T3 polymerase promoter site. Second strand cDNA is synthesized as in the Eberwine protocol. The RNA transcripts produced in this second amplification round are oriented in the sense direction. Modified figure from Kaposi-Novak . (). ( and ). An oligo dT primer and a terminal continuation (TC) primer containing a T7 promoter sequence in the sense oriented transcription are added to the mRNA sample for first strand cDNA synthesis. TC is based on the observation of the reverse transcriptase enzyme adds a few Cs and also Gs nonspecifically at the end of mRNA templates. The TC primer anneals with this stretch and provides a binding site for second strand cDNA synthesis. RNA transcription can be driven using a promoter sequence attached to either the 3′ or the 5′ oligo primers and in thus generates either sense or antisense RNA transcripts. For further methodological details of the terminal continuation strategy see Che and Ginsberg (). () The first and subsequent rounds of amplification follow the same procedure as the classical Eberwine method. The final aRNA is reverse transcribed into sense cDNA and used as a template for Klenow labeling, yielding fluorescently labeled antisense cDNA, which are in the correct orientation for hybridization to oligo arrays. Adapted figure from Schlingemann . ()