Obtaining high-quality, intact RNA is the first and often the most critical step in performing many fundamental molecular
biology experiments. Most RNA isolation products use the powerful chaotropic salt solution guanidinium isothiocyanate for
sample lysis and homogenization, followed by organic extraction and alcohol precipitation or solid-phase purification. Organic
extraction using acidified phenol and chloroform removes proteins, lipids, and DNA from the RNA sample, which is then recovered
by alcohol precipitation. Solid-phase, column-based procedures utilize glass-fiber filters that bind RNA; proteins and DNA
are removed by washing them through the filter. RNA is then eluted from the filter with RNase-free water. An alternative to
column-based procedures is magnetic beads, which also bind RNA very efficiently under selective conditions.
Usually the first step after RNA isolation is to measure how much was recovered — the yield. There are also several methods
for assessing RNA integrity and purity, both components of RNA quality, that may affect downstream applications. The following
sections discuss aspects of measuring quantity and quality of isolated RNA.
Figure 1. Total RNA Yield (µg) from Different Tissues (mg) * g total RNA/mg tissue. This is intended as a general guide only
and may vary depending on physiological state or organism.
CALCULATING RNA YIELDS
RNA yields vary widely depending on tissue or cell type, physiological state at the time of tissue removal, method of tissue
disruption, and efficiency of RNA recovery (i.e., the RNA isolation method used, and the proficiency of the researcher performing
the test). Figures 1 and 2 provide information about estimated yields of RNA (total + messenger RNA [mRNA]) from different
Figure 2. Yield of Total and mRNA from Different Tissues and Cells *Actual values may vary depending on tissue or cell type,
physiological state, etc.
UV spectroscopy is a commonly used and easy method for quantification of RNA. The absorbance of a diluted RNA sample is measured
at wavelengths of 260 nm and 280 nm. The nucleic acid concentration is calculated using the Beer-Lambert law, which predicts
a linear change in absorbance with concentration. It is simple to perform, and UV spectrophotometers are available in most
laboratories. Some of the more recently developed spectrophotometers (e.g., the NanoDrop ND-3300 Fluorospectrometer) make
it possible to use only nanoliter to microliter volumes of sample.
Removing DNA Interference Because UV spectroscopy does not discriminate between RNA and DNA, it is advisable to first treat RNA samples with RNase-free
DNase to remove contaminating DNA. Contaminants such as residual proteins and phenol can also interfere with absorbance readings,
so care must be taken to remove them during purification steps following RNA isolation.
Factors Affecting Absorbance The A260/A280 ratio is dependent on both pH and ionic strength. As pH increases, the A280 decreases while the A260 is unaffected, resulting in an increasing A260/A280 ratio.1 Water often has an acidic pH, it can lower the A260/A280 ratio. The remedy is to use a buffered solution with a slightly alkaline pH — such as Tris-EDTA (TE) at pH 8.0 — as a diluent,
and as a blank to ensure accurate and reproducible readings.