NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation.

Author(s): Eikmans M, Rekers NV, Anholts JD, Heidt S, Claas FH

Publication: Blood, 2013, Vol. 121, Page e81-9

PubMed ID: 23327925 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of storage and thawing of peripheral blood mononuclear cells (PBMC) prior to RNA extraction, RNA extraction and reverse transcription method and RNA degradation on RNA yield and integrity and mRNA and microRNA (miRNA) levels from PBMC and peripheral blood lymphocyte blasts (PBL).

Conclusion of Paper

Compared to RNA obtained from fresh PBMC, RNA from PBMC stored at -20 degrees C in RNAlater or Trizol before extraction was of significantly lower quality as determined by RNA integrity number (RIN), but freezing PBMC in dimethylsulfoxide (DMSO) had no effect on RIN, regardless of thaw method. While storage of PBMC before or after freezing in RNAlater versus Trizol had no effect on RIN, the forkhead box P3 (FoxP3) expression levels, as determined by real-time qRT-PCR, were significantly higher when PBMC were stored before or after freezing in Trizol than RNAlater. RNA yield was not affected by RNA extraction method from PBMC or PBL (5 methods compared), and RIN were comparable between most methods, but RIN were significantly higher for RNA isolated from PBMC by RNA-Bee than RNeasy. While RNA extraction method from PBL had no effect on mRNA levels, levels of beta-actin and transforming growth factor beta 1 (TGF-beta 1) were highest when RNA from PBMC was extracted using RNeasy, followed by NucleoSpin and lowest when extracted with mirVana. Similarly, miRNA levels were not affected by extraction method from PBL, but levels of miR-142-5p were higher in RNA isolated from PBMC with the mirVana kit than the NuceloSpin kit (only two kits considered). The highest real time qRT-PCR signals from PBL or PBMC RNA were obtained when RNA was reverse transcribed with SuperScript III, followed by BioScript or RevertAid, with the lowest by AMV-RT, regardless of RNA input or transcript. As RIN declined, real-time qRT-PCR signal intensity declined, but relative mRNA expression remained constant until samples reached a RIN of <4.2. In contrast, miRNA levels, with the exception of SNORD49 which declined at RIN of 4.7 or lower, were stable. Loss of the real-time qRT-PCR signal with mRNA degradation occurred regardless of the amplicon size, but the loss of signal occurred more rapidly with increased amplicon size.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of storing PBMC in RNAlater or Trizol for 6 days at -20 degrees C or in DMSO for 3 days at -80 degrees, thawing method, and frozen storage of cells in RNAlater or Trizol after thawing on RNA yield and integrity as well as quantification of FoxP3 mRNA. RNA was either extracted immediately from PBMC or PBMC were stored frozen in RNAlater or Trizol at -20 degrees C for 6 days or in DMSO at -80 degrees C for 3 days. After thawing in a 37 degrees C water bath, cells were transferred drop-wise to room temperature 10% fetal calf serum in culture media (standard thawing) or 50% fetal calf serum in culture media with Benzonase (Benzonase thawing). After thawing, RNA was extracted immediately, or cells were stored at -20 degrees C in RNAlater or Trizol for 6 days.

    Summary of Findings:

    Compared to RNA obtained from fresh PBMC, RNA from PBMC stored at -20 degrees C in RNAlater or Trizol before extraction had significantly lower RNA quality as determined by RIN (p<0.05) and nonsignificantly lower FoxP3 expression, as determined by real-time qRT-PCR, but freezing cells in DMSO had no effect on RIN, regardless of thaw method. While storage before or after freezing in RNAlater versus Trizol had no effect on RIN, the FoxP3 real-time qRT-PCR signals were significantly higher when storage before or after freezing was in Trizol than RNAlater (p<0.05, both).

    Biospecimens
    Preservative Types
    • RNAlater
    • Frozen
    • None (Fresh)
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    RNA Automated electrophoresis/Bioanalyzer
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Frozen
    RNAlater
    None (fresh)
    Storage Thaw temperature/condition 10% fetal calf serum in culture media
    50% fetal calf serum in culture media with Benzonase
    Storage Short-term storage solution RNAlater
    DMSO
    Trizol
    Storage Storage temperature -20 degrees C
    -80 degrees C
    Storage Storage duration 0 days
    3 days
    6 days
    Real-time qRT-PCR Specific Targeted nucleic acid FoxP3
  2. Study Purpose

    The purpose of this study was to determine the effects of RNA extraction method, reverse transcription method, and heat-induced RNA degradation on RNA yield and integrity as well as mRNA and miRNA expression. PBMC were obtained from 3 patients. RNA yield and RIN numbers from PBMC were compared for all 5 extraction methods, but mRNA expression was limited those extracted with RNeasy, mirVana or NucleSpin, and miRNA expression was limited to RNA obtained with mirVana or NucleSpin. Lymphocyte blasts were obtained from the PBMC of 4 patients after 7 days of culture, RNA was extracted using all 5 kits, and mRNA and miRNA expression was determined using specimens extracted with each of the 5 kits. miRvana-extracted RNA from PBL was used to investigate the effects of reverse transcription method. To determine the effect of RNA degradation, RNA from PBL and PBMC was stored at 90 degrees C for up to 3 h.

    Summary of Findings:

    RNA yield was not affected by RNA extraction method from PBMC (5 methods compared), and while RIN were comparable between most methods, they were significantly higher for RNA isolated by RNA-Bee than RNeasy (p<0.05). Further, levels of beta-actin and TGF-beta 1 were highest when RNA was extracted from PBMC using RNeasy, followed by NucleoSpin and lowest when extracted with mirVana (RNA-Bee and Trizol were not considered). Levels of miR-142-5p and miR223 were higher in RNA isolated from PBMC with the mirVana kit than the NuceloSpin kit (only two kits considered), but only the difference in miR142-5p was significant. Levels of miR-155 and FoxP3 in PBMC were unaffected by RNA extraction method. RNA extraction method from PBL did not affect RNA yield, RIN, or levels of mRNA and miRNA measured by real-time qRT-PCR. The highest real time qRT-PCR signals in RNA from PBL or PBMC were obtained with SuperScript III, followed by BioScript or RevertAid, with the lowest by AMV-RT, regardless of RNA input amount or transcript. RNA was increasingly degraded with increasing storage at 90 degrees C. As RIN declined, real-time qRT-PCR signals for mRNA declined, but relative mRNA expression remained constant until RIN of <4.2 at which point the ratio of cell division cycle 25 (CDC25) to beta-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was significantly higher (p<0.05). In contrast, with the exception of SNORD49 which declined at RIN of 4.7, miRNA levels were stable, and thus relative miRNA levels were also unaffected by RIN. Loss of the real-time qRT-PCR signal with mRNA degradation occurred regardless of the amplicon size, but the loss of signal occurred more rapidly with increased amplicon size.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    RNA Real-time qRT-PCR
    RNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Aliquots and Components Blood and blood products Peripheral blood mononuclear cells
    Lymphocyte
    Analyte Extraction and Purification Analyte isolation method RNA-Bee
    Trizol
    RNeasy
    mirVana
    NucleSpin
    Real-time qRT-PCR Specific Targeted nucleic acid Beta-actin
    GAPDH
    TGF-beta1
    FoxP3
    cdc25
    HPRT-1
    SNORD38B
    SNORD49A
    miR-103
    miR-142-5p
    miR-155
    miR-223
    miR-191
    miR-26b
    Real-time qRT-PCR Specific Length of gene fragment 45-415 bp
    Real-time qRT-PCR Specific Template/input amount 100 ng RNA
    500 ng RNA
    1500 ng RNA
    Storage Storage duration 0 min
    15 min
    30 min
    45 min
    1 h
    1.25 h
    1.5 h
    2 h
    2.5 h
    3 h
    Real-time qRT-PCR Specific Technology platform Reverse transcribed with SuperScript III
    Reverse transcribed with BioScript
    Reverse transcribed with RevertAid
    Reverse transcribed with AMV-RT

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