technote_eco_absolutequant_sybr_green.pdf

Technical Note: Real-Time PCR Introduction Gene expression is the process by which genetic information is converted into a functional product. This process uses an intermedi- ate molecule, RNA, which is transcribed from DNA and then used as a template to translate the message into a protein product. Studies of gene expression provide a window into how an organism’s genetic makeup enables it to function and respond to its environment. Real-Time PCR can be used to quantify gene expression by two methods: relative and absolute quantifi cation. The relative quantifi ca- tion method compares the gene expression of one sample to that of another sample: drug-treated samples to an untreated control, for example, using a reference gene for normalization. Absolute quantifi - cation is based on a standard curve, which is prepared from samples of known template concentration. The concentration of any unknown sample can then be determined by simple interpolation of its PCR signal (Cq) into this standard curve. Purpose This Protocol provides a step-by-step guide for quantifying the level of gene expression of a gene of interest using the absolute quantifi cation method in the Eco Real-Time PCR System. The steps covered in this protocol include: RNA Extraction and Quantifi cation1. cDNA Synthesis2. Preparation of Serial Dilutions3. Real-Time PCR Amplifi cation4. Data Analysis5. Several of these steps are prone to variability that can lead to data inconsistancies. For more detailed discussion of these issues, refer to the Nature Protocol by Nolan, Hands, and Bustin (1). Step 1: RNA Extraction and Quantifi cation Optimal quantifi cation of gene expression requires high quality, intact RNA. This implies appropriate sample collection and disruption, as well as proper isolation and storage of RNA. If you already have puri- fi ed and quantifi ed RNA, go directly to Step 2. Once total RNA has been purifi ed it needs to be quantifi ed. UV spec- troscopy is the tradit