Impact of Sample Processing and Storage Conditions on RNA Quality of Fresh-Frozen Cancer Tissues.
Author(s): Chen YY, Han QY, Chen QY, Zhou WJ, Zhang JG, Zhang X, Lin A
Publication: Biopreserv Biobank, 2023, Vol. , Page
PubMed ID: 37040277 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA integrity numbers (RIN) among RNA extracted from 210 specimens of different tumor types (colon, rectal, gastric, liver and lung), tumor stages, and durations of frozen storage (2-166 months). RIN and RNA purity were also compared between case-matched specimens homogenized manually and using a TissueLyser-32L and among frozen specimens that experienced a power outage and were permitted to reach different temperatures.
Conclusion of Paper
RIN values were comparable among the tumor types examined. RIN values ≥7 were obtained from a higher percentage of stage III-IV tumor specimens than stage I-II tumor specimens. Specimens that were stored at -80℃ for longer than 5 y had a higher percentage of specimens with a RIN <7 than those with a RIN ≥7. RIN and RNA purity (ratio of the absorbance at 260 nm to 280 nm) were comparable when tissues were homogenized using a TissueLyser-32L instrument or manually with mortar and pestle. RIN and purity were also comparable among specimens that were maintained at a temperature ≤0℃ and those stored continuously at -80℃; however, RIN and RNA purity were much lower when RNA was extracted from tumor specimens subjected to 4 h at room temperature.
Studies
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Study Purpose
This study compared RIN among RNA extracted from 210 specimens of different tumor types (colon, rectal, gastric, liver and lung), tumor stages, and durations of frozen storage (2-166 months). RIN and RNA purity were also compared between case-matched tumor specimens that were homogenized manually with a mortar and pestle or using a TissueLyser-32L instrument, and among tumor aliquots that were permitted to reach different temperatures after a freezer power outage. A total of 210 tissues were collected during surgical resection from patients with colon cancer, rectal cancer, gastric cancer, liver cancer and lung cancer (35 each). The tissues were snap-frozen in liquid nitrogen and stored for 2-166 months at -80℃. Unless otherwise specified, tissues were homogenized in TRIzol reagent with a TissueLyser-32L instrument and steel beads. RNA was extracted using TRIzol reagent and quantified by NanoDrop. RNA integrity was assessed using the RNA 6000 Nano Kit on a bioanalyzer. The effects of tissue type and storage duration were investigated using all specimens. To investigate the effects of tissue homogenization method, case-matched segments of 18 frozen gastric cancer tissue specimens were homogenized in TRIzol reagent with a TissueLyser-32L instrument and steel beads or manually using a mortar and pestle and liquid nitrogen before the addition of Trizol. To investigate the effect of frozen storage temperature fluctuations, specimens from 10 colorectal cancer patients were aliquoted, snap-frozen in liquid nitrogen, and stored at -80℃. The freezer power was terminated and aliquots of each tissue were removed when the temperature reached -80℃ -60℃, -40℃, -20℃ and 0℃ and 4 h after reaching room temperature and then and stored at -80℃ until extraction and analysis. RNA was extracted with the phenol-chloroform based TRIzol reagent and quantified by NanoDrop. RNA integrity was assessed using the RNA 6000 Nano Kit on a bioanalyzer.
Summary of Findings:
RIN values were comparable among the tissue types evaluated. A higher percentage of stage III-IV tumor specimens had RIN values ≥7 than stage I-II tumor specimens (70.1% versus 29.9%, P=0.027). There was no effect of frozen storage duration on RIN when analyzed by the number of months or years of storage. When frozen storage was longer than 5 years, there was a higher percentage of specimens with a RIN <7 than those with a RIN ≥7 (76.7% versus 60.5%, P=0.048). RIN and RNA purity (ratio of the absorbance at 260 nm to 280 nm) were comparable when the tissue was homogenized using either a TissueLyser-32L instrument or manually with a mortar and pestle. RIN and RNA purity were also comparable among specimens maintained ≤0℃ and those stored continuously at -80℃; however, RIN and RNA purity were much lower when RNA was extracted from tumor specimens subjected to 4 h at room temperature.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Prognostic factor Stage I-II
Stage III-IV
Storage Thaw temperature/condition Stored continuously at -80℃
Stored in unplugged -80℃ freezer until it reached -60℃
Stored in unplugged -80℃ freezer until it reached -40℃
Stored in unplugged -80℃ freezer until it reached -20℃
Stored in unplugged -80℃ freezer until it reached 0℃
Stored in unplugged -80℃ freezer until it reached room temperature for 4 h
Storage Storage duration 2-166 months
<5 years
>5 years
Biospecimen Acquisition Biospecimen location Colon
Rectum
Stomach
Lung
Liver