Two-Step RT-qPCR mixes - SYBR Green
The two-step RT-qPCR involves two distinct reactions, starting with first strand cDNA synthesis (reverse transcription-RT) and then amplifying part of the resulting cDNA by quantitative PCR in a separate tube . Therefore, two-step RT-qPCR is useful for detecting multiple genes in a single RNA sample. The separation of the RT and quantitative PCR reactions makes it possible to optimize the reaction conditions for each step as well as the flexibility with priming by reverse transcription (oligo (dT) primers, random hexamers or gene-specific primers) and PCR DNA polymerase and PCR components). Compared to one-step RT-qPCR, the disadvantages of two-step RT-qPCR include several steps for extended workflow, additional handling and processing of the sample, and increase the risk of contamination and variation results.
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