Maximizing PCR and RT-PCR Success

20 PCR Brochure 10/2016 Figure 23. Highly sensitive MSP amplification. Human genomic DNA was purified from blood using the QIAamp DNA Blood Mini Kit, and various amounts (1 ng – 1 μg) were converted using the EpiTect Bisulfite Kit. PCR was performed using the HotStarTaq Plus Master Mix Kit and two sets of primers designed to amplify converted DNA. 5 μl of each product was loaded onto a 1.3% agarose gel. The HotStarTaq Plus Master Mix Kit allowed specific amplification from all DNA concentrations. M: marker; C: negative control. 1 µ g 1 µ g 1 0 0 n g 1 0 0 n g 1 0 n g 1 0 n g 1 n g 1 n g M C C M – 707 bp – 150 bp Viral research with one-step RT-PCR RNA secondary structure can affect RT-PCR results in a number of ways. During reverse transcription, regions of RNA with complex secondary structure can cause the reverse transcriptase to stop or dissociate from the RNA template. Truncated cDNAs that do not include the downstream primer-binding site are not amplified during PCR. In some cases, the reverse transcriptase skips looped structures, resulting in deletions in the cDNA which lead to truncated PCR products. When amplifying a low-abundance transcript or viral sequences, these problems are even more critical. A well-balanced system, consisting of reverse transcriptase, a stringent hot-start enzyme, and an optimized buffer system is crucial for applications such as viral detection or gene expression analysis, where maximum sensitivity is often required (Figure 24). Download our specialized protocol for one-step RT-PCR for viral samples. The buffer provided with the QIAGEN OneStep RT-PCR Kit and QIAGEN OneStep Ahead RT-PCR Kit allows reverse transcription to be performed at an elevated temperature (50˚C). This high reaction temperature improves the efficiency of the reverse transcriptase reaction by disrupting secondary structures and is particularly important for one-step RT-PCR performed with limiting template amounts. copies M 2 2 , 0 0 0 2 2 0 0 2 2 0 2 2 2 Figure 24. Efficient detection of viral RNA. A 336 bp fragment of F-gene mRNA was reverse-transcribed and amplified from Sendai virus RNA isolated from persistently infected Vero cells. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit and the indicated number of viral genome copies.* M: markers. * Data kindly provided by H. Rausch, Max Planck Institute for Biochemistry, Martinsried, Germany as part of the project “Experimental control of virological work at safety levels 2 and 3 in Bavaria,” supported by the Bavarian Ministry of the Environment.

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