When preparing high quality RNA, it is critical to rapidly inactivate endogenous ribonucleases. Traditionally, tissue samples have either been snap-frozen in liquid nitrogen and then stored at 80°C, or immediately processed in an RNA lysis buffer. Freezing provides the convenience of being able to process the tissue at a later date.
Frozen tissue must be ground to a powder while still frozen, and then placed into RNA lysis buffer (usually guanidinium, lithium or SDS based), at which point it is homogenized. However, many researchers allow their tissue to thaw before processing, and end up with RNA that is not fully intact. We therefore decided to investigate the effect on RNA integrity of thawing frozen samples prior to homogenization.
Replicate mouse kidney samples were flash-frozen in liquid nitrogen and stored at 80°C. One sample was thawed to room temperature prior to RNA isolation and then processed for total RNA isolation. The second sample was immediately processed for total RNA isolation. The resulting RNA samples were quantitated and 2 µg of each prep was loaded onto a 1% formaldehyde-agarose gel, (Figure 1B) transferred to a nylon membrane for Northern blotting, and hybridized with a high specific activity RNA probe for ß-actin (Figure 1C). The resulting bands were quantitated on a phosphorimager. Each sample (100 ng) was also analyzed on an Agilent 2100 bioanalyzer (Figure 1A).
Figure 1. Effect of Tissue Freeze/Thaw on RNA Quality. A. Each sample (100 ng) was analyzed on the Agilent 2100 bioanalyzer. The 28S rRNA:18S rRNA ratio in the thawed sample was less than that of the frozen control, indicating RNA degradation in the thawed sample. B. Each total RNA sample (2 µg) was analyzed by gel electrophoresis on a 1% formaldehyde-agarose gel in the presence of 0.5 µg/ml ethidium bromide. C. The RNA from Panel B was transferred to a positively charged nylon membrane for Northern blotting. The membrance was hybridized with 106 cpm/ml of a high specific activity RNA probe for mouse ß-actin.
Clearly there is more degradation in the RNA isolated from the thawed kidney sample as compared to the control. This experiment was repeated with several different tissues (data not shown) and a similar result was observed. It is known that the freeze-thaw process can disrupt cellular compartments where RNases are sequestered, giving them access to the RNA. These experiments show that it is necessary to keep tissue frozen at all times prior to homogenization it in an RNA lysis buffer in order to keep the RNA intact.