Titan™ One Tube RT-PCR System Protocol & Troubleshooting

Product No. 11855476001

Protocol

Amount of poly(A) RNA template

For best results, start with 1 ng - 100 ng poly(A)-RNA.

Template preparation

The required template RNA can be isolated using the mRNA Capture Kit or the mRNA Isolation Kit.

Control reactions

Perform the RT-PCR without the RT step. Meaning after setup, directly perform the denaturation step of PCR (2 min at +94 °C) to inactivate the reverse transcriptase. Alternatively, you can treat the RNA template with RNaseA before starting the denaturing step of PCR. No PCR product should be visible when the reaction is complete. If a PCR product is produced, the following are possible sources:

1. There is DNA contamination in the RNA template.
2. DNA contamination in the reaction mixture is present.
3. PCR product results of RNA template, since the reverse transcriptase had enough time to transcribe during the heating phase of the denaturation step.

Multiplex RT-PCR

The Titan™ One Step RT-PCR System is ideally suited for single-tube multiplex RT-PCR applications, such as gene expression assays which require normalization by comparison to a control (housekeeping) gene. Amplification of the gene(s) of interest simultaneously with the control gene in a single-tube reaction reduces the risk of introducing assay-to-assay variances which could influence the interpretation of results. An example of such single-tube duplex amplification of target and housekeeping gene can be found in Biochemica newsletter 1997, No 4: Optimization of the RT-PCR Method Using the Titan™ One Tube RT-PCR System.

Other publications describing this kind of application are:

1. M. R. Hynd, H. L. Scott and P. R. Dodd (2001). GlutamateNMDA receptor NR1 subunit mRNA expression in Alzheimer′s disease. Journal of Neurochemistry, Vol. 78, No. 1, 175-182.
2. C. K. Ward, S. R. Lumbley, J. L. Latimer, L. D. Cope, and E. J. Hansen (1998). Haemophilus ducreyi Secretes a Filamentous Hemagglutinin-Like Protein. Journal of Bacteriology, Vol. 180, No. 22, 6013-6022
3. Y. Liu and M. F. Schneider (1998). Fibre type-specific gene expression activated by chronic electrical stimulation of adult mouse skeletal muscle fibres in culture.The Journal of Physiology, 512.2, 337-344.
4. D. A. Lewis, M. K. Stevens, J. L. Latimer, C. K. Ward, K. Deng, R. Blick, S. R. Lumbley, C. A. Ison, and E. J. Hansen (2001). Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems. Infection and Immunity, Vol. 69, No. 9, 5626-5634.
5. B. Li, L. A. Nolte, J.-S. Ju, D. H. Han, T. Coleman, J. O. Holloszy and C. F. Semenkovich (2000). Skeletal muscle respiratory uncoupling prevents diet-induced obesity and insulin resistance in mice. Nature Medicine Vol. 6, No. 10, 1115 - 1120.
   

Troubleshooting

To exclude artifacts from DNA targets like residual genomic DNA contaminations from RNA preparations (see also under primer design), or contaminating DNA from previous amplifications appropriate positive and negative control reactions should be included.

1. To control contaminating DNA amplicons from previous PCR always set up a control reaction without RNA template. In this reaction no PCR product should be visible.

2. a) Set up a control reaction where the RT reaction is not performed by

• using instead of the Titan™ enzyme mix, Expand™ enzyme mix (from Expand™ High Fidelity or Expand™ Long Template) or Taq DNA polymerase (2.5 U) or alternatively
• heat the Master Mix 2 for 2 minutes at +94 °C (to inactivate reverse transcriptase AMV), than follow step 1 in the protocol and proceed with step 3 (do not use step 7). Be always aware that even Taq DNA polymerase has a low reverse transcriptase activity.
  

b) Treat your RNA preparation with RNase A then proceed with the RT-PCR protocol.

EXPAND is a trademark of Roche
TITAN is a trademark of Roche