Northern blotting, nuclease protection assays and RT-PCR are frequently used to analyze gene expression. While qualitative results from these assays are sometimes sufficient, most researchers require quantitative results.

Quantitative data can be expressed in either relative or absolute terms. For relative quantitation, RNA levels of the gene under investigation are compared from sample to sample using an internal control to normalize for differences in sample concentration and loading. Ideally, the internal control should be a gene expressed at a constant level across the sample set. In absolute quantitation, signal intensities representing the expression levels of the experimental gene are compared to a standard curve generated by samples containing a concentration titration of an external standard.

In this technical bulletin, we describe relative and absolute quantitation applied to Northern blotting, nuclease protection assays and RT-PCR. We also discuss how to choose an internal control for relative quantitation.

Relative Quantitation

In relative quantitation, RNA levels of the gene of interest are compared from sample to sample using an internal control to normalize for differences in sample concentration and loading. As the internal control should be expressed at a constant level across all samples, both choosing and authenticating this control are critical to successful relative quantitation. In the paragraphs below, we describe commonly used internal controls and how these are authenticated.

Internal Standard RNA Probes
Inconsistencies in RNA isolation and in the commonly used RNA analysis procedures listed above can introduce errors into the analysis process. One method for minimizing these errors is to simultaneously measure a cellular RNA that has a constant expression level between samples. This RNA serves as an internal standard or reference against which other RNA values can be normalized. This process corrects for sample to sample variation.

The ideal RNA species for an "internal standard" should be expressed at a constant level across all of the types of samples being analyzed. For example, the internal standard should be expressed equally among different tissues of an organism, at all stages of development, and for both control and experimentally treated cell types. A constituitively expressed "housekeeping" gene would appear to be a good model for an internal standard RNA. Unfortunately, there is no one single RNA with a constant expression level in all of these situations (although 18S rRNA appears to come close to being an ideal internal control under the broadest range of experimental conditions, see below). It is therefore necessary to identify the appropriate control RNA for the particular set of experimental RNA samples to be studied.

ß-actin
ß-actin mRNA was one of the first RNAs used as an internal standard. It encodes a ubiquitous cytoskeleton protein and is expressed at moderately abundant levels. In a 10 µg sample of total RNA, there is approximately 300 pg of ß-actin mRNA, representing 0.1% of mRNA or 0.003% of total RNA. This is compared to 0.3–3 pg of a rare mRNA species. The ß-actin gene is highly conserved in eukaryotes (1) and expressed in most cell types (2). However, the level of expression has been shown to vary in some tissues, including cultured adipocytes (3), mammary epithelial cell lines, breast fibroblast cell lines (4), and cultured human colon carcinoma cells (5). Experiments at Ambion have shown ß-actin mRNA levels to be high in normal mouse spleen, brain, embryo, heart, hypothalamus and kidney and relatively lower in mouse liver, lung and muscle (Figure 1).

Figure 1. Expression Level of Several Common Internal Standards Compared across Mouse Tissues Using a Ribonuclease Protection Assay. High specific activity antisense RNA probes (1.15 x 109 cpm/µg) to GAPDH, ß-actin and cyclophilin were synthesized from Ambion's pTRI-GAPDH, pTRI-beta-actin, and pTRI-cyclophilin mouse DNA templates, respectively, in a MAXIscript in vitro transcription reaction. 4 x 104 cpm of each probe was added to 5 µg total RNA from different mouse tissues and processed in a multiple probe ribonuclease protection assay using the RPA II Kit. Results show a comparison of the relative expression levels of each probe within a given tissue and across mouse tissues. Control lanes of yeast RNA and probe +/- RNase are shown on the left and Ambion's Century Markers provide molecular weight standards on the right.