Stability analysis of liver cancer-related microRNAs.
Author(s): Li Y, Jiang Z, Xu L, Yao H, Guo J, Ding X
Publication: Acta Biochim Biophys Sin (Shanghai), 2011, Vol. 43, Page 69-78
PubMed ID: 21173058 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of heating serum before miRNA extraction and the effects of storing extracted miRNA or serum at different temperatures, durations, pH values, and freeze-thaw cycles on levels of liver-specific miRNA. The resistance of extracted miRNA and miRNA in serum to digestion with RNAse A and DNAse I, and the interindividual and gender variability of liver-specific miRNA levels were also investigated.
Conclusion of Paper
Incubating serum at 75°C for 5 min prior to RNA extraction resulted in lower average cycle threshold (CT) values for both miR-221 and miR-222. Levels of miR-25, miR-221, or miR-222 were not affected by storage of serum or extracted RNA at -80°C, -20°C, 4°C, 37°C for 3 h, at room temperature for up to 24 h, for 3 h at 37°C in a variety of pH values (1-13), or through 10 freeze-thaw cycles, but following these treatments a >50% reduction in 18s ribosomal (r)RNA levels was observed. While miR-25, miR-221, and miR-222 in serum were resistant to digestion with RNAse A or DNAse I at 37°C for 12 h, a 30-80% reduction in miR-25, miR-221, and miR-222 levels for liver RNA under the same conditions. Very strong correlations in serum miRNA levels were observed between individuals (R=0.91-0.99). There was no effect of patient gender on levels of miR-25, miR-221, or miR-222.
Studies
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Study Purpose
The purpose of this study was to determine effects of heating serum before miRNA extraction, the effects of storing serum or extracted miRNA at a variety of temperatures (-80°C, -20°C, 4°C, 37°C), durations, pH values (1-13), and freeze-thaw cycles on levels of liver-specific miRNA and the resistance of miRNA to digestion with RNAse A and DNAse I. Tumor tissue and serum from an unspecified number of patients with liver cancer and serum from 22 healthy individuals were stored at -80°C prior to analysis. RNA was extracted from 25 mg of frozen liver sections using Norgen’s animal tissue RNA purification kit. RNA was extracted from serum using Trizol. The number of specimens used for each experiment was not specified.
Summary of Findings:
Incubating serum at 75°C for 5 min prior to RNA extraction resulted in lower average CT values for both miR-221 and miR-222. Storage of serum or miRNA extracted from liver at -80°C, -20°C, 4°C, 37°C for 3 h, at room temperature for up to 24 h, for 3 h at 37°C in a variety of pH values (1-13), or through 10 freeze-thaw cycles did not affect levels of miR-25, miR-221, or miR-222, but led to a >50% reduction in 18s rRNA. While miR-25, miR-221, and miR-222 in serum were resistant to RNAse A or DNAse I digestion at 37°C for 12 h, a 30-80% reduction in miR-25, miR-221, and miR-222 levels was observed when RNA from liver was digested with DNAse I or RNAse A at 37°C for 12 h. Very strong correlations in serum miR-levels were observed between each of the 22 healthy individuals (R=0.91-0.99) and there was no effect of patient gender on miRNA levels.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Liver
Serum
Preaquisition Patient gender Male
Female
Storage Storage temperature -80°C
-20°C
4°C
Room temperature
37°C
Storage Storage duration 1 h
3 h
6 h
12 h
24 h
Storage Freeze/thaw cycling 0 cycles
2 cycles
5 cycles
7 cycles
10 cycles
Analyte Extraction and Purification Nucleic acid digestion No RNAse A
3 h of RNAse A
6 h of RNAse A
12 h of RNAse A
No DNAse I
3 h of DNAse I
6 h of DNAse I
12 h of DNAse I
Analyte Extraction and Purification Analyte isolation method Preheat for 5 min at 75°C before extraction
No preheating