RT-qPCR preparation

When the RNA has been extracted from the sample and reverse transcribed into cDNA you are ready to quantify the transcripts with RT-qPCR.

The transcript can only be quantified relative to a reference gene. The reference genes are usually genes essential for the cell because they are needed for basic cellular functions. Reference genes are expected to be expressed at relatively constant levels in most of the cells of an organism under normal and diseased conditions.

Choose a reference gene and design primer pairs specific for your target and reference gene.

Before you start with the qPCR experiment you need to produce master mixes for each of your primer pairs. Use exactly the same concentration of primers and add the qPCR reagent.

The qPCR reagent contains: - SYBR-Green, which fluoresces when bound to double stranded DNA. - DNA polymerase to amplify the DNA.

Mix your cDNA samples with each master mix and start the qPCR experiment.