Real-time QRT-PCR is a powerful tool for mRNA quantitation in biological samples. Alien QRT-PCR Inhibitor Alert is a high-quality external control for detecting inhibitors in RNA samples for quantitative RT-PCR experiments. The Alien RNA transcript is an in vitro-transcribed RNA molecule that has no significant homology to any known nucleic acids.
The inhibitor alert test is performed by comparing amplification of a known amount of Alien RNA in the absence and presence of the experimental RNA samples of interest. Specifically, the threshold cycle, or Ct, for amplification of the Alien RNA is measured in reference reactions containing the Alien RNA transcript alone. In separate inhibitor test reactions, each of the RNA samples of interest are added to an Alien RNA amplification reaction. An increased Ct for the Alien RNA in a specific test reaction, relative to the reference reactions, indicates the potential presence of one or more inhibitors of QRT-PCR.
The Alien QRT-PCR inhibitor alert can be used to detect inhibitors in either 1-Step (single-tube) or 2-Step (two-tube) QRT-PCR assays that employ SYBR® Green dye for detection. Separate protocols are provided for 1-Step and 2-Step QRT-PCR applications.
The Alien QRT-PCR inhibitor alert is also well suited for assay standardization applications. Using the Alien QRT-PCR inhibitor alert as a reference control to generate standard curves allows data comparisons from multiple experiments, across platforms, and between laboratories. The Alien RNA is produced in large lots and subjected to stringent quality-control measures to ensure the availability of consistent reference RNA material over long-term experimental studies.
Properties of the Alien RNA Transcript and Optimized Primers
The Alien RNA transcript is a ~500-nt, polyadenylated RNA molecule that is synthesized by in vitro transcription. The Alien RNA transcript does not have significant homology to any known nucleic acid sequences currently in public databases, as determined by BLAST comparisons against NIH sequence databases. In addition to its unique sequence, the Alien RNA transcript was designed with a GC content of approximately 50% and is predicted to have little secondary structure. The RNA has been extensively tested in QRT-PCR assays, in combination with the optimized primers provided, to ensure optimal QRT-PCR amplification efficiency, thus allowing maximum sensitivity for inhibitor detection. The Alien RNA transcript is free of contaminating DNA.
An optimized primer pair for amplification of the Alien RNA target is provided. The primer pair has been optimized for QRT-PCR efficiency, sensitivity, and specificity. Using the reaction conditions specified in the product manaul (Protocols Section), amplification is highly specific for the expected 239-bp amplicon, with very little primer-dimer formation.
For Research Use Only. Not for use in diagnostic procedures