Storage of purified RNA at sub-zero temperatures (usually –20ºC or –80ºC) is a time-tested method for ensuring stability of the entire RNA population without bias. Recently, some researchers have questioned whether chemical instability or RNA adherence to tube surfaces skews representational levels of individual miRNA species during the first few days after RNA isolation and sub-zero storage. Here we show that no such bias is introduced in RNA samples (total RNA, including miRNA) prepared with the mirVana™ miRNA Isolation Kit.
Experimental Design: RNA Preparation & Storage
Powdered stocks of frozen mouse liver and brain tissue were created by pulverizing frozen tissue under liquid nitrogen with a pre-chilled mortar and pestle. Lysates representing the first step of the mirVana miRNA Isolation Kit protocol were made from these stocks by adding 1 volume of tissue to 10 volumes of mirVana Lysis/Binding Solution and immediately homogenizing with a motorized Rotor Stator homogenizer (e.g., Polytron® homogenizer). Each tissue lysate was divided into 600 µL aliquots, flash frozen using liquid nitrogen, and stored at –80ºC for a minimum of 1 hour to ensure that all samples went through the same freeze/thaw cycle. This process creates a stable, homogeneous substrate for extraction of equivalent RNA over time, through the action of both the RNase-inactivating denaturants in the lysis solution and the cold storage.
The tissue lysate aliquots were thawed and RNA was isolated per the mirVana miRNA Isolation Kit protocol. Eluted RNA was divided between commercially available stick and nonstick microfuge tubes, and stored at either –20ºC or –80ºC for time points between 1 hr and 15 days.
Results and Conclusions: Stability of miRNA Shown by Real-Time RT-PCR
MicroRNA expression was measured with triplicate TaqMan® MicroRNA Assays for 5 miRNA targets: Let-7d, miR-16, miR-32, miR-335, and miR-451. No significant differences in CT values were observed between the different RNA storage time points (Figure 1, liver) or storage conditions (Figure 2, liver and brain). Nonstick tubes showed no clear benefit over regular tubes (all data shown are from regular tubes, like those included in the mirVana miRNA Isolation Kit).
Figure 1. miRNA Expression Over Time From RNA Samples Stored at –80°C. RNA was purified and frozen at –80°C for 1 hour to 15 days. Frozen RNA samples were thawed simultaneously and single RT reactions were run for each sample using the Applied Biosystems TaqMan® MicroRNA Reverse Transcription Kit, the appropriate TaqMan miRNA primer, and 10 ng total RNA. Triplicate PCR reactions were run on the Applied Biosystems 7900HT Fast Real-Time PCR System with 1.33 µL of each RT reaction and the respective TaqMan MicroRNA Assay. CT values were averaged, and the standard deviation was calculated.
When samples were stored at –80ºC, miRNA degradation was not apparent in either liver (Figure 1) or brain (data not shown) lysates. Similar CT values were also observed for samples divided and stored at –20ºC and –80ºC (Figure 2), indicating that it is safe to store miRNA extracted using the mirVana miRNA Isolation Kit at either –20ºC or –80ºC.
Figure 2. miRNA Expression Comparison Between Storage Conditions. RNA samples were extracted on the first day, divided, frozen in liquid nitrogen, and stored at the indicated temperature. On day 15, the samples were thawed simultaneously and single RT reactions were run for each sample using the Applied Biosystems TaqMan® MicroRNA Reverse Transcription Kit, the appropriate TaqMan microRNA primer, and 10 ng total RNA. Triplicate PCR reactions were run on the Applied Biosystems 7900HT Fast Real-Time PCR System with 1.33 µL of each RT reaction and the respective TaqMan MicroRNA Assay. CT values were averaged, and the standard deviation was calculated.
Yvonne Potucek and Rick Conrad • Applied Biosystems, Austin, TX