Application: Quantitative RT-PCR is used to quantify mRNA in both relative and absolute terms. It can be applied for the quantification of mRNA expressed from endogenous genes, and transfected genes of either stable or transient transfection. It is the most sensitive method as yet in quantitative analysis of mRNA. Principle: PCR amplification follows the formula:
A = B(1+e)n
A=amplified products, B=input templates, n=cycle number, and e=amplification efficiency. Factors affecting amplification efficiency in the RT-PCR process include the efficiency of reverse transcription, Mg 2+ / dNTPs/ primer concentrations, enzyme activity, pH, annealing temperature, cycle number, temperature variation, tube to tube variation etc. Since PCR results in a million fold amplification, variation in any of the above factors during the amplification process will significantly affect the final output; therefore routine RT-PCR can not be used for the purpose of quantitative analysis. This elimination, however, can be overcome using quantitative RT-PCR(1). This method uses an external template as the internal control for all the steps in RT-PCR process. The quantitative RT-PCR follows the formula:
A = B (1+e)n
A' B'(1+e)n
A=amplified products, B= input templates, A'=amplified control products, and B'=input control templates. Any effect on the amplification efficiency will equally affect both templates, thus providing a linear relationship between both wild type and control templates in the amplification process. The output ratio between the two templates directly reflects the input ratio between these two templates.
As the amount of input control templates is known, the amount of wild type templates in the RNA sample can be easily determined.
The system described below has been developed for the quantification of endogenous expression from either single or multiple genes. The expression from several genes can be simultaneously quantified in both relative and absolute terms. In comparison to other RNA quantification methods, this assay is highly sensitive, highly quantitative, and gives cleaner results when performed properly. RT-PCR can be used for analyzing expression from single or multiple genes, and analyzing changing expression patterns in diseases etc.
Procedure:
1. RNA Preparation
A number of procedures are available for preparation of RNA from tissue culture cells or mononuclear cells. The NP-40 lysis method affords a good choice (5) for preparation of RNA from tissues or primary cells, however, guanidine method is better(6). From our experience, a commercial product Trizol solution (GIBCOBRL), a modified guanidine solution, gives the best result. For isolation of RNA from white blood cells, first use PMN solution (Robbions Scientific Corporation) to isolate white blood cells which will include all mononuclear and polymorphonuclear cells. The isolated RNA should be treated with DNase I to remove genomic DNA contamination. Quantify the RNA by measuring O.D260 and check the quality on an agarose gel. Typically, the RNA yield from a 20 ml blood sample is about 20 mg.
Grow cells in exponential phase. If desired, activate the gene with conditions such as heat shock, etc. Prepare RNA for the analysis of endogenous gene expression or for the analysis of a stable transfected gene (for studying the expression of a transient transfected gene, you will need to co-transfect the wild type plasmid with a control plasmid containing the modified homologous gene controlled by a constitutive promoter, in order to control the transfection efficiency (2, 3).
During RNA isolation and analysis, it is essential to maintain an RNase free environment. All solutions should be RNase free. Always wear gloves. Keep samples on ice and immediately freeze RNA samples after using them (4).
The following procedure is a modified version of the NP-40 RNA extraction method for tissue culture cell lines (5). It is simple, fast and gives high yield of high quality RNA.
