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Multimodal Analysis of Circulating Cell-free RNA (ccfRNA), Circulating Cell-free DNA (ccfDNA) and Genomic DNA (gDNA) From Blood Samples Collected in PAXgene Blood ccfDNA Tubes1Andrea Ullius, 1Eric Provencher and 1Thorsten Voss 1PreAnalytiX GmbH, Hombrechtikon, Switzerland Please contact [email protected] for further questions or visit www.preanalytix.com
IntroductionWhile circulating cell-free DNA (ccfDNA) from blood is widely used as an analyte in liquid biopsy research applications, circulating cell-free RNA (ccfRNA) has recently gained relevance for biomarker studies. Combined insights from both analytes promise to increase the understanding of underlying molecular processes. However, challenges in the preanalytical workflow, such as choosing blood collection tubes and extraction methods suitable for multimodal analysis, must still be overcome.
We investigated multimodal extraction and analyses of ccfRNA, ccfDNA and gDNA from one blood sample collected using the PAXgene® Blood ccfDNA Tube (RUO)*†.
PAXgene Blood ccfDNA Tube
Data from www.preanalytix.com
The PAXgene Blood ccfDNA Tubes (RUO)* enable:• Stabilization of ccfDNA for 10 days up to 25°C, 7 days up to 30°C and 3 days up to 37°C• Single tube collection, stabilization, transport and storage• Standardized preanalytical sample processing
PAXgene Blood ccfDNA stabilization reagent helps prevent release of gDNA into plasma
Results
MethodsWhole blood samples were collected from healthy consented donors into PAXgene Blood ccfDNA Tubes* (PreAnalytiX®), BD Vacutainer® K2EDTA tubes (BD), Cell-Free DNA BCT® (Streck®), RNA Complete BCT™ (Streck) and LBgard® Blood Tubes (Biomatrica®). Plasma was generated by double centrifugation immediately after blood collection or after storage for up to three days. Cell-free nucleic acids were extracted as shown in the workflow.
ccfRNA yield in plasma after blood storage in EDTA and PAXgene Blood ccfDNA tubes
• Quantitative PCR analysis revealed comparable yields of miRNA, mRNA and ccfDNA targets from plasma of blood collected in PAXgene Blood ccfDNA Tubes and EDTA tubes
• After blood storage in PAXgene Blood ccfDNA Tubes* for up to 3 days, RNA targets (both intra- and extravesicular extracted with exoRNeasy and miRNeasy, respectively) could still be detected with improved stabilization over EDTA.
10
15
20
25
30miR451
miRNeasy(n = 6)
mRNA ccfDNA
10
15
20
25
30 let7a
exoRNeasy(n = 14)
EDTA PAXgeneEDTA PAXgeneEDTA PAXgeneEDTA PAXgene10
15
20
25
30
18S rDNAQIAamp circ. NA
(n = 6)
15
20
25
30
35
ACTB mRNAQIAamp circ. NA
(n = 6)
10
15
20
25
30miR150
exoRNeasy(n = 14)
miRNA
EDTA PAXgene
CTv
alue
CTv
alue
CTv
alue
CTv
alue
CTv
alue
A Comparison at TTP0
CT values of qPCR analysis of miR150, let7a and miR451 micro RNAs, ACTB mRNA and 18S rDNA (ccfDNA) in PAXgene and EDTA plasma, generated directly after blood collection (test time point = TTP0) and extracted using the indicated kit.
miRNA mRNA
05
10152025406080
100
ACTB mRNAQIAamp circ. NA Kit
(n = 4)
0
5
10
15
20
25
miR451 miRNeasy(n = 4)
0
5
10
15
20
25
let7a exoRNeasy(n = 4)
0
5
10
15
20
25
miR150 exoRNeasy(n = 4)
Fold
cha
nge
rela
tive
to T
TP0
Fold
cha
nge
rela
tive
to T
TP0
Fold
cha
nge
rela
tive
to T
TP0
Fold
cha
nge
rela
tive
to T
TP0
B Relative fold change upon whole blood storage
EDTA T1d
EDTA T3d
PAXgene
T1d
PAXgene
T3d
EDTA T1d
EDTA T3d
PAXgene
T1d
PAXgene
T3d
EDTA T1d
EDTA T3d
PAXgene
T1d
PAXgene
T3d
EDTA T6d
PAXgene
T6d
EDTA T1d
EDTA T3d
PAXgene
T1d
PAXgene
T3d
Calculated fold change (relative to TTP0) of qPCR analysis of miR150, let7a, miR451 micro RNAs and ACTB mRNA of PAXgene and EDTA plasma, generated after 1, 3 or 6 days of whole blood storage. RNA was extracted using the indicated kit.
miRNA yield in plasma after blood storage in stabilization tubes
• RNA extraction and detection sensitivity was impacted by blood collection tubes containing formaldehyde-releasing formulations (Streck and Biomatrica) as indicated by slightly higher CT values at TTP0 and lower RNA stabilization efficiency after 3 days of storage.
miRNA
PAXgene
Streck
cfDNA
Streck
RNA
Biomatr
ica
PAXgene
Streck
cfDNA
Streck
RNA
Biomatr
ica
PAXgene
Streck
cfDNA
Streck
RNA
Biomatr
ica20
25
30
35
40
miR451miRNeasy
(n = 6)
15
20
25
30
35
let7aexoRNeasy
(n = 6)
10
15
20
25
30
miR150exoRNeasy (n = 6)
CTv
alue
CTv
alue
CTv
alue
A Comparison at TTP0
CT values of qPCR analysis of miR150, let7a and miR451 micro RNAs in PAXgene, Streck cfDNA, Streck RNA and Biomatrica plasma, generated directly after blood collection (TTP0) and extracted using the indicated kit.
miRNA ccfDNA
Fold
cha
nge
rela
tive
to T
TP0
Fold
cha
nge
rela
tive
to T
TP0
Fold
cha
nge
rela
tive
to T
TP0
Fold
cha
nge
rela
tive
to T
TP0
EDTA T3d
PAXgene
T3d
Streck
cfDNA T3d
Biomatr
ica T3d
0123456
18S rDNAQIAamp circ. NA
(n = 6)
Streck
RNA T3d
Biomatr
ica T3d
02468
10
50100150
miR451miRNeasy
(n = 6)
02468
10
50100150
let7aexoRNeasy
(n = 6)
02468
101214161820
miR150exoRNeasy
(n = 6)
B Relative fold change upon whole blood storage
Streck
RNA T3d
Streck
cfDNA T3d
PAXgene
T3d
Streck
RNA T3d
Biomatr
ica T3d
Streck
cfDNA T3d
PAXgene
T3d
Streck
RNA T3d
Biomatr
ica T3d
Streck
cfDNA T3d
PAXgene
T3d
Calculated fold change (relative to TTP0) of qPCR analysis of miR150, let7a, miR451 micro RNAs and 18S rDNA in PAXgene, Streck cfDNA, Streck RNA and Biomatrica plasma, generated after 3 days of storage (T3d). RNA was extracted using the indicated kit.
Genomic DNA yield and integrity
• PAXgene Blood ccfDNA Tubes* enabled efficient gDNA extraction from residual blood cells after plasma separation following 3 days of whole blood storage with intact DNA as indicated by stable DNA integrity index
• gDNA yield and integrity were reduced by collection and storage in Streck RNA and Biomatrica tubes
gDNA
6
7
8
9
10DNA integrity index
(n = 11)
DIN
0
50
100
150
200
250gDNA concentration
(n = 11)
gDN
A c
onc.
(µg/
ml)
PAXgene
T0
PAXgene
T3d
Streck
cfDNA T0
Streck
cfDNA T3d
Streck
RNA T0
Streck
RNA T3d
Biomatr
ica T0
Biomatr
ica T3d
PAXgene
T0
PAXgene
T3d
Streck
cfDNA T0
Streck
cfDNA T3d
Streck
RNA T0
Streck
RNA T3d
Biomatr
ica T0
Biomatr
ica T3d
Concentration and DNA integrity index (DIN) assessment of gDNA extracted from whole blood in PAXgene Blood ccfDNA Tubes*, Streck cfDNA, Streck RNA and Biomatrica tubes. DNA was extracted from cellular fraction after first plasma centrifugation step using the QIAamp Blood DNA Kit and analyzed with the Agilent Genomic DNA ScreenTape® on the TapeStation System.
Multimodal workflow for the analysis of ccfRNA, ccfDNA and gDNA
1900 x g15 min
• BD Vacutainer K2EDTA tubes• PAXgene Blood ccfDNA Tube*• Streck Cell-Free DNA BCT• Streck RNA Complete BCT• Biomatrica LBgard Blood Tube
• Whole blood storage for up to 3 days at room temperature• 1st centrifugation: According to manufacturer’s recommendation / ISO standard• 2nd centrifugation:10 min, 3,000 g / 2,800 g (Streck RNA)
• QIAGEN exoRNeasy Midi Kit (intravesicular ccfRNA)• QIAGEN miRNeasy Serum/Plasma Kit (total plasma ccfRNA)• QIAGEN QIAamp Circulating Nucleic Acid Kit (total plasma ccfDNA and ccfRNA)
• QIAGEN QIAamp Blood DNA Mini Kit
• (cDNA synthesis)• PCR analysis
• Multimodal analysis from one blood sample:- ccfDNA- ccfRNA- gDNA
Bloodcollection +stabilization
Bloodstorage +
centrifugation
ccfRNA + ccfDNA
extraction from plasma
gDNAextraction
from cellular fraction
(RT-)PCR analysis Insight
1,900 x g15 min
Conclusions• The non-crosslinking technology of the PAXgene Blood ccfDNA Tube* enables the isolation and analyses of cell-free miRNA, mRNA, ccfDNA and cellular gDNA.• Along with recent data on CTC enrichment and detection**, RNA data from this study show that the PAXgene Blood ccfDNA Tube* can be used as part of a multimodal workflow in liquid biopsy research.• Other blood collection tubes for ccfDNA stabilization showed impaired analysis efficiency after whole blood storage for the tested miRNA targets and gDNA.
Disclaimer*The PAXgene Blood ccfDNA Tube is For Research Use Only in the US. Not for use in diagnostic procedures. **Babayan et al. 2020 (Poster shown at AACR Meeting: Advances in Liquid Biopsies, Miami, FL)†For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN or PreAnalytiX handbook or user manual. QIAGEN and PreAnalytiX handbooks and user manuals are available at www.qiagen.com or www.preanalytix.com or can be requested from QIAGEN Technical Services or your local distributor.
Trademarks are the property of their respective owners.
© 2020 PreAnalytiX GmbH. PreAnalytiX, the PreAnalytiX Logo and all other trademarks are property of PreAnalytiX GmbH, Hombrechtikon, CH.
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AACR Virtual Annual Meeting II, 2020
