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
Conclusion of Paper
Studies
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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:
Analyte Technology Platform RNA Spectrophotometry RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(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
-
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:
Analyte Technology Platform RNA Spectrophotometry RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(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