'Future-proofing' blood processing for measurement of circulating microRNAs in samples from biobanks and prospective clinical trials.
Author(s): Murray MJ, Watson HL, Ward DM, Bailey S, Ferraresso M, Nicholson JC, Gnanapragasam VJ, Thomas B, Scarpini CG, Coleman N
Publication: Cancer Epidemiol Biomarkers Prev, 2017, Vol. , Page
PubMed ID: 29254935 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of blood collection tube type, extraction method, and centrifugation delays on levels of miRNA in plasma and serum. The effects of centrifugation delays and tube type on hemolysis were also investigated using different assessment methods.
Conclusion of Paper
miRNA levels were highest in serum followed by EDTA plasma and Streck DNA plasma. The miRNA levels were much lower in specimens collected in Streck RNA tubes and were not improved by changes in the extraction method. Although hemolysis increased with pre-centrifugation storage of blood in EDTA or Streck DNA tubes and was detected using real-time PCR in almost all specimens regardless of centrifugation delay, hemolysis as determined by visual inspection and by spectrophotometry was only detected in specimens stored for at least 7 days in Streck DNA tubes or 1 h in EDTA tubes. Use of a second centrifugation step or a high speed centrifugation step to obtain EDTA plasma reduced the recovery of miRNA. Use of the Norgen plasma/serum RNA purification kit reduced the variability in miRNA quantification compared to when extraction was performed with the Qiagen miRNeasy serum/plasma kit, but the CT values were still higher for plasma than for serum and were not improved by the addition of a proteinase K step.
Studies
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Study Purpose
This study compared levels of six microRNAs in plasma from blood collected in EDTA tubes, Streck DNA tubes, and Streck RNA tubes with levels found in serum. Blood was obtained from five patients with testicular malignant germ cell tumors (GCTs) and two patients with non-GCT testicular malignancies into Sarstedt S-Monovette Z-Gel (serum), Sarstedt S-Monovette K3E (EDTA plasma), Streck Cell-Free DNA BCT, and Streck Cell-Free RNA BCT. Serum was obtained after 30 min clot time by centrifugation at 3000 x g for 10 min. EDTA plasma was obtained from Sarstedt K3EDTA tubes by centrifugation of blood at 1600 x g for 10 min followed by recentrifugation of the supernatant at 14,400 x g for 10 min. Plasma was obtained from both Streck tubes by centrifugation at 300 x g for 20 min followed by at 5000 x g for 10 min. All plasma and serum were stored at -80֯C until analysis. RNA was extracted using miRNeasy serum/plasma kit with QIAzol.
Summary of Findings:
Levels of miR-30b-5p, miR-30c-5p, and miR-191-5p were highest in serum followed by EDTA plasma and Streck DNA plasma and were lowest in plasma from Streck RNA tubes. While the standard deviations in miRNA levels were generally low, they were higher in specimens collected in Streck RNA tubes than in other plasma types or in serum. Levels of the hemolysis markers of miR-23a-3p and miR-451a were also highest in serum specimens followed by EDTA plasma, Streck DNA plasma, and finally Streck RNA plasma. Importantly, based on visual inspection and the absorbance at 414 nm, specimens collected in Streck DNA plasma tubes were not considered hemolyzed while those collected in EDTA plasma were hemolyzed. Plasma from specimens in both EDTA and Streck DNA tubes was considered hemolyzed using the Delta CT value (CT of miR-23a-3p – miR-451a), but the value was higher in plasma from EDTA tubes.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck DNA plasma tube
Streck RNA plasma tube
EDTA tube
Gel Separator serum tube
Spectrophotometry Specific Technology platform Real-time PCR
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Study Purpose
This study investigated the effects of delayed centrifugation of blood collected in EDTA and Streck DNA tubes on hemolysis using two specimens. Blood was collected from two healthy patients and a patient with Leydig cell tumor into Streck DNA tubes and Sarstedt S-Monovette K3E (EDTA) tubes and stored at room temperature for 0, 2 4, 7, 10, and 14 days before centrifugation. EDTA plasma was obtained by centrifugation of blood at 1600 x g for 10 min followed by recentrifugation of the supernatant at 14,400 x g for 10 min. Plasma was obtained from Streck DNA tubes by centrifugation at 300 x g for 20 min followed by at 5000 x g for 10 min. All plasma and serum were stored at -80֯C until analysis. RNA was extracted using miRNeasy serum/plasma kit with QIAzol.
Summary of Findings:
Hemolysis was first visually apparent in specimens collected in Streck DNA tubes when they were stored for 7 days or in EDTA tubes after 1 h. Using a threshold of absorbance of 414 nm >0.2 units, hemolysis was detected by spectrophotometric analysis in all EDTA plasma but the level of hemolysis increased significantly when centrifugation was delayed for 4 days or more. In contrast, hemolysis only occurred in specimens from Streck DNA tubes when centrifugation was delayed by 7 days or more. Interestingly, hemolysis was evident in both EDTA plasma and Streck DNA plasma using a threshold of an 8-cycle difference between miR-23a-3p and miR-451a, regardless of centrifugation delay. In contrast, only one of seven serum specimens was considered hemolyzed when centrifuged within 1 h, but this difference in detection was attributed mostly to lower levels of the housekeeping gene in plasma.
Levels of all six housekeeping miRNA (miR-30b-5p, miR-30c-5p, miR-191-5p, miR-26a-5p, miR-130b-3p, and miR-146a-5p) investigated increased with increasing delays to centrifugation. However, normalization of the levels of the housekeeping miRNA to the red blood cell marker miR-451a attenuated the effects of delayed centrifugation, indicating that the changes were attributable to hemolysis.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform Protein Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Acquisition Type of collection container/solution Streck DNA plasma tube
EDTA tube
Gel Separator serum tube
Storage Storage duration 1 h
2 days
4 days
7 days
10 days
14 days
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Study Purpose
This study investigated the effects of RNA extraction techniques on miRNA quantification. Blood was collected in Sarstedt S-Monovette K3E (EDTA plasma) tubes from five patients with testicular malignant GCTs, two patients with non-GCT testicular malignancies, two healthy patients, and one patient with Leydig cell tumor. EDTA plasma was obtained by centrifugation of blood at 1600 x g for 10 min followed by recentrifugation of the supernatant at 14,400 x g for 10 min and stored at -80֯C until analysis. RNA was extracted using the miRNeasy serum/plasma kit, the Norgen plasma/serum RNA purification kit, and the mirVana PARIS isolation kit. To eliminate protein interference, some specimens were digested with Proteinase K for 1 h at 60֯C. miRNA were quantified by TaqMan real-time PCR.
Summary of Findings:
Use of the Norgen plasma/serum RNA purification kit reduced the variability in miRNA quantification compared to when extraction was performed with the Qiagen miRNeasy serum/plasma kit but the CT values were 2-5 cycles higher in plasma extracted using the Norgen kit than for serum extracted with the Qiagen kit. Inclusion of a proteinase K step in the Norgen extraction did not improve miRNA recovery. Use of the mirVana PARIS kit to extract RNA from Streck RNA tubes resulted in CT values that were >2 cycles higher than when RNA was extracted with the miRNeasy kit.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Norgen plasma/serum RNA isolation kit
Qiagen miRNeasy serum/plasma kit
mirVANA Paris kit
Biospecimen Acquisition Type of collection container/solution Streck DNA plasma tube
Streck RNA plasma tube
EDTA tube
Gel Separator serum tube
Analyte Extraction and Purification Protein digestion Proteinase K for 1 h
None
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Study Purpose
The purpose of this study was to compare different methods to measure hemolysis. Blood was collected in Sarstedt S-Monovette K3E (EDTA plasma) tubes from five patients with testicular malignant GCTs, two patients with non-GCT testicular malignancies, two healthy patients, and one patient with Leydig cell tumor. EDTA plasma was obtained by centrifugation of blood at 1600 x g for 10 min followed by recentrifugation of the supernatant at 14,400 x g for 10 min and stored at -80֯C until analysis. RNA was extracted using the miRNeasy serum/plasma kit. miRNAs were quantified by TaqMan real-time PCR and hemolysis was determined by spectrophotometry.
Summary of Findings:
Only 20 of 32 specimens (62%) were considered hemolyzed by spectrophotometry using a cut-off of an absorbance at 414 nm of >0.2, but 30 of 32 (94%) were considered hemolyzed based on the delta CT value (CT of miR-23a-3p minus miR-451a). Specimens classified as hemolyzed by spectrophotometer had higher delta CT values (P=0.009) and lower CT values for miR-451a (P<0.0001). There was a strong nonlinear association between the absorbance at 414 nm and the CT value for miR-451a (R2=0.818) but only a weak association with the delta CT (R2=0.224). Importantly, the dynamic range of absorbance at 414 nm was greater than that for CT values making it a more valuable tool for assessment of hemolysis.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Benign
- Neoplastic - Germ Cell
- Normal
Platform:
Analyte Technology Platform Protein Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Spectrophotometry Specific Technology platform Real-time PCR
-
Study Purpose
The purpose of this study was to investigate the impact of centrifugation speed on miRNA values in plasma. Blood was collected from a single patient with Leydig cell tumor into Sarstedt S-Monovette K3E (EDTA) tubes and stored at room temperature for 0, 2 4, 7, 10, and 14 days before centrifugation. EDTA plasma was obtained by centrifugation of blood at 1600 x g for 10 min and then some of the supernatant was recentrifuged at 14,400 x g for 10 min. All plasma and serum were stored at -80֯C until analysis. miRNAs were quantified by real-time PCR. The effects of centrifugation speed were also investigated using data from published studies from breast cancer patients.
Summary of Findings:
The mean expression level of all five miRNAs together or independently were significantly higher when a single centrifugation at 1600 x g was used to isolate EDTA plasma instead of a dual centrifugation (1600 x g and then at 14,400 x g)(P=0.03). Similarly, EDTA plasma isolated by a single centrifugation had higher levels of all five miRNAs than serum (P=0.01). Investigation of published global miRNA profiling studies revealed mean miRNA levels and individual levels were higher in specimens subjected to low speed centrifugation (1000 or 2000 x g) than those that were centrifuged at 10,000 x g (P<0.001, both), but miRNA levels were comparable in specimens centrifuged at 1000 x g and 2000 x g. In particular, levels of the housekeeping miR-30b-5p, mIR-30c-5p, and 191-5p were particularly affected. Although levels of miRNA were almost always lower when specimens were subjected to high speed centrifugation, the magnitude of the difference ranged from 0 to 8 cycles depending on the miRNA in question.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Multiple speeds compared