RNAlater®
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To demonstrate the utility of RNAlater for preserving proteins and RNA for long periods of time, RNA and protein were isolated from mouse tissue that had been stored for more than a year in RNAlater. Fresh brain and kidney tissues were dissected and immediately submerged in 5 volumes of RNAlater, incubated at 4°C overnight and then transferred to -20°C. Thirteen months later, the RNAlater treated samples were thawed at room temperature, about 50 mg of tissue was collected, and the remainder of the samples were refrozen. Small tissue pieces from both tissues were subjected to the standard PARIS procedure to quickly isolate total RNA and protein. The samples were disrupted with a motorized homogenizer in 400 µl of Cell Disruption Buffer. The bulk of the resulting lysate (300 µl) was then immediately mixed with 1 volume of 2X Lysis/Binding Solution for RNA isolation using the glass fiber filter provided with the PARIS Kit, and the remainder of the lysate was reserved for protein analysis.
Figure 1. RNA and Protein from Tissues Stored in RNAlater® for >1 Year. RNA and protein were isolated with the PARIS™ Kit from ~50 mg of mouse brain or kidney stored for 13 months at -20°C in RNAlater. Purified RNA (1 µg) was analyzed on a denaturing agarose gel and subsequently transferred, blotted, and hybridized using the NorthernMax®-Gly Kit (Ambion). A portion of the protein fraction (10 µg) was used for Western blot analysis with antibodies specific for GAPDH or Hur proteins.
Figure 2. RNA and Protein Isolation from Stabilized Samples. Isolation from ~30 mg of mouse liver, brain or kidney 5 days after collection. Samples were either kept frozen at -80ºC, or stored in RNAlater® at 4ºC.
As shown in Figure 1, no obvious RNA degradation was observed by denaturing agarose gel analysis or by Northern blot hybridized with probes specific for U1 snRNA, or ß-actin and GAPDH mRNAs. Analysis of the protein fraction by Western blot showed that the recovered proteins were full-length. (Note: The multiple bands observed in the Hur blot correspond to brain-specific higher molecular weight isoforms of the Hur protein.) Figure 2 further confirms that protein yield and quality are not affected by RNAlater treatment. These results demonstrate that RNAlater is an efficient sample collection/stabilization reagent for protecting RNA, and that RNAlater-treated samples can be used for routine protein analysis such as Western blotting.
It is especially useful for the following applications:
Figure 1. RNA and Protein from Tissues Stored in RNAlater® for >1 Year. RNA and protein were isolated with the PARIS™ Kit from ~50 mg of mouse brain or kidney stored for 13 months at -20°C in RNAlater. Purified RNA (1 µg) was analyzed on a denaturing agarose gel and subsequently transferred, blotted, and hybridized using the NorthernMax®-Gly Kit (Ambion). A portion of the protein fraction (10 µg) was used for Western blot analysis with antibodies specific for GAPDH or Hur proteins.
Figure 2. RNA and Protein Isolation from Stabilized Samples. Isolation from ~30 mg of mouse liver, brain or kidney 5 days after collection. Samples were either kept frozen at -80ºC, or stored in RNAlater® at 4ºC.
As shown in Figure 1, no obvious RNA degradation was observed by denaturing agarose gel analysis or by Northern blot hybridized with probes specific for U1 snRNA, or ß-actin and GAPDH mRNAs. Analysis of the protein fraction by Western blot showed that the recovered proteins were full-length. (Note: The multiple bands observed in the Hur blot correspond to brain-specific higher molecular weight isoforms of the Hur protein.) Figure 2 further confirms that protein yield and quality are not affected by RNAlater treatment. These results demonstrate that RNAlater is an efficient sample collection/stabilization reagent for protecting RNA, and that RNAlater-treated samples can be used for routine protein analysis such as Western blotting.
It is especially useful for the following applications:
- Archiving samples for future analysis
- Collecting samples at different time points without having to process them immediately
- Shipping samples without dry ice
- Collecting samples in the field
