October 1, 2005 (Vol. 25, No. 17)
RNA Isolation and Validation
Obtaining high-quality RNA is perhaps the most critical step in many molecular biology experiments, including RT-PCR, microarrays, Northern analysis, nuclease protection assays, RNA mapping, in vitro translation, and cDNA library construction.
Successful RNA purification requires rapid processing and attentive handling of samples prior to isolation. Since endogenous RNases are released after harvesting, it is essential to immediately inactivate these RNases to prevent RNA degradation. Although endogenous RNase activity varies dramatically across different tissues (Figure 1), it must be controlled to enable the recovery of intact RNA.
One way to do this is by freezing samples in liquid nitrogen. Flash frozen tissues must be ground or pulverized at cryogenic temperatures prior to homogenization, which is laborious and time consuming. Yet, RNA can still degrade if the dissection takes too long or if the frozen tissue thaws.
An alternative is to place samples in an RNA stabilization solution like RNAlater from Ambion (www. ambion.com) that protects the RNA in intact, unfrozen samples. Figure 2 shows the reservation of total RNA by denaturing agarose gel electrophoresis and Northern analysis of tissues stored in RNAlater.
Thorough tissue disruption is essential for high RNA quality and yield. RNA that is trapped in intact cells is removed with cellular debris and is unavailable for subsequent isolation. Disruption is usually achieved by mechanical methods or by grinding the tissue. Physical dissociation of tissue, however, is cumbersome, low-throughput, and requires cleaning of equipment between samples. Open tube mechanical homogenization can also present biohazards through aerosols released from the sample.
The novel Multi-Enzymatic Liquefaction of Tissue (MELT) Total Nucleic Acid System from Ambion is a simpler and safer alternative. This technology enables hands-free tissue lysis through the use of powerful catabolic enzymes and a potent small molecule RNase inhibitor. RNA integrity obtained using the MELT system was comparable to that obtained using two leading manufacturers’ isolation kits: 1) single reagent, phenol-based lysis solution, followed by glass fiber filter (GFF) treatment, and 2) a guanidinium isothiocyanate (GITC), GFF-based method. (Figure 3a).
However, RNA yields from the MELT protocols were up to three times greater (Figure 3b), in part due to closed tube disruption, which limits losses from tissue handling.
Unlike conventional RNA purification reagents, MELT proteases irreversibly inactive RNases by digesting them to peptide fragments. Figure 4 shows that intact RNA can be purified from MELT lysates even after 10 days of storage at room temperature while RNA from a GITC-based lysate was highly degraded within three days of room temperature storage.
RNA isolation kits typically use organic extraction and alcohol precipitation, or solid-phase purification after sample homogenization. The former rely on acidified phenol and chloroform to remove proteins, lipids, and bulk DNA from the RNA sample, which is then recovered by alcohol precipitation.
Column-based procedures utilize GFF that bind RNA and DNA, while cellular contaminants are removed by washing the filter. DNA is eliminated by digestion with DNase I. RNA is then eluted in RNase-free water.
Magnetic beads are an alternative to traditional RNA isolation methods and offer more consistent RNA recoveries. Another benefit of magnetic beads is the elimination of filter clogging due to cellular particulates in samples. Moreover, since only a small volume of magnetic beads is enough for high efficiency binding, the bound RNA can be eluted in low volumes of elution buffer.
Intact RNA is essential for many techniques used in gene expression studies especially Northern analysis, cDNA library construction, and cDNA labeling for microarray analysis. Quantitative RT-PCR and nuclease protection assays both involve analysis of smaller regions of RNA and are therefore more tolerant of partially degraded RNA. Regardless of the downstream application, it is important to assess RNA integrity before gene expression analysis.
One method measures the ratio of the absorbance of the RNA at 260 nm versus 280 nm. Highly pure RNA has a ratio of >1.8, whereas a lower ratio suggests contamination by cellular proteins.
Though simple and easy to perform, A260/280 ratio does not provide any information about RNA integrity.
28S/185 rRNA Ratio
Another method compares the intensity of the 28S and 18S ribosomal RNA (rRNA) bands after electrophoresis. Intact RNA will have sharp 28S and 18S rRNA bands while smeared bands indicate degraded RNA. Bands can be assessed on agarose gels, or by microcapillary electrophoresis using the Agilent 2100 bioanalyzer. While a ratio of 1.52.0 (28S:18S rRNA) indicates intact RNA, the ratio can vary with the tissue type.
The RNA Integrity Number (RIN)
The RIN is a new software tool designed by Agilent Technologies to estimate the integrity of total RNA samples. RNA intactness is determined by the appearance of RNA species along the entire electrophoretic trace. The algorithm assigns a score of 1 to 10, where a RIN of 10 RNA is completely intact. This automated calculation from the electropherogram makes universal and unbiased sample comparisons possible.
Obtaining high-quality RNA starts even before isolation. Biosamples must be stored in a stabilization agent to protect the RNA and maintain global representation. Closed tube enzymatic cell disruption can provide efficient RNA release and irreversible RNase degradation. Magnetic bead adsorption of RNA efficiently separates RNA from other cellular components. New instruments, like the bioanalyzer, and new standards like the RIN, provide more effective ways to assess RNA quality.