Using Universal Reference RNA for Microarray and RT-PCR Experiments

As with any biological assay, microarray and RT-PCR experiments require optimization before performing the experiment of interest and obtaining publication-quality results. The repeated tweaking of parameters and conditions unnecessarily expends difficult to obtain RNA samples needed for the desired experiment. Other RNA samples can meet these needs instead. Commercial sources of universal reference RNA contain a broad, almost genome-wide, representation of genes and are of sufficient quality for standard gene expression profiling methods. As a result, universal reference RNA not only helps conserve more precious experimental RNA samples but also streamlines and improves single- or two-color microarray as well as real-time or conventional (end-point) RT-PCR experiments. This article further outlines the advantages of using universal reference RNA as well as how to use it for these four different applications.

Normalization Control for Two-Color Microarrays:

The most useful application of universal reference RNA involves two-color fluorescent microarray applications. This method allows direct comparison of gene expression between two different samples by labeling them with two different fluorescent dyes and hybridizing them to the same microarray. However, more complicated experiments involving multiple pair wise comparisons between several samples require an internal control, preferably one for every gene represented by the microarray, to compare any sample to any other sample. Universal reference RNA fulfils this requirement when labeled with one dye and hybridized to the same microarray as each experimental RNA sample labeled with the other dye. In this way, the signal for each gene can be normalized to its own unique factor allowing comparisons of gene expression across multiple samples.

To perform a two-color microarray experiment with universal reference RNA, convert the experimental RNA obtained from each control or treated cells or tissue to probe labeled with one dye (the green Cy3 dye for example). Convert a large sample of universal reference RNA to probe labeled with another dye (the red Cy5 dye for example). Allow one Cy3 probe and an aliquot of the Cy5 probe to hybridize to the same microarray, and then detect both colored signals. For each gene, normalize the value from the experimental RNA sample to the value from the universal reference RNA (divide the former Cy3 signal by the latter Cy5 signal). Compare these ratios between microarrays to calculate fold changes in gene expression between each experimental condition. This use of universal reference RNA for two-color microarray experiments applies to the available methods for both the direct and indirect sample labeling. However, before beginning such a study using direct incorporation, consider pilot experiments using opposing color assignments for the experimental samples and the universal RNA (or a so-called dye swapping experiment). The different rates of incorporation of the two different dyes have been known to skew gene expression profiling results.

Standardization and Troubleshooting for Single Color Microarrays:

Only a few microarray platforms support the fluorescent detection of two different dyes. Many others, referred to here as single-color microarrays, only accommodate the detection of one fluorescent, chemiluminescent, or radioactive signal. When performing single-color (or even two-color) microarray experiments for the first time, several conditions or parameters should be optimized before committing precious experimental samples to the methodology including but not limited to: RNA isolation and quality, labeled target or probe synthesis, hybridization and detection. High RNA quality is perhaps the most important and most tedious of these conditions to achieve particularly for the uninitiated.

Commercial universal reference RNA is of high enough quality to guarantee the successful synthesis of labeled probe for hybridization to a microarray. In this way, other experimental parameters involved in hybridization and detection can be optimized without wasting more precious experimental RNA samples and without waiting to hone one's RNA isolation technique. Alternatively, if a routine microarray experiment suddenly yields substandard results, universal reference RNA helps determine whether poor experimental RNA quality may have been at fault.

To optimize or troubleshoot a single- or two-color microarray experiment using universal reference RNA, simply synthesize labeled probe using the universal reference RNA and follow the protocol for hybridizing it to the microarray and detecting the hybridized probe. Adjust other conditions (such as the hybridization and detection steps) to achieve reliable and detectable signals from a majority of the spots (genes) on the microarray without saturating too many of the signals.