Reference gene stability in Peripheral Blood Mononuclear Cells
determined by qPCR and NanoString
Lars Radke1,2, Christoph Giese3, Annika Lubitz3, Stephan Hinderlich4, Grit Sandig4,
Michael Hummel2 and Marcus Frohme1
1University
of Applied Sciences Wildau (15745) GERMANY, 2Charité Berlin (12203)
GERMANY, 3ProBioGen AG Berlin (13086) GERMANY, 4Beuth University of Applied
Sciences Berlin (13353) GERMANY
MIQE checklist
(E): Essential Information (D): Desired Information
Experimental design
Definition of experimental and control groups (E)
12 Samples in two experimental set-ups:
6 samples from experiment with PBMC
6 samples from experiment with PBMC+mDC
In each subset 1 sample is unstimulated (NC), 1 sample is stimulated with LPS (PC) and 4
samples are stimulated with vaccine preparations from derived from CHO cells (three different
concentrations) or SF9 cells (1 concentration) (for further information see M&M part).
Number within each group (E)
Due to limited amounts of vaccine and high costs of cell culturing in a special reactor (human
artifical lymph node reactor) sample material is limited. Therefore, the number within each group is
one.
Assay carried out by the core or investigator’s laboratory? (D)
Vaccine preparations were generated at the Beuth University of Applied Sciences, Berlin and at
the ProBioGen AG, Berlin.
Cell Culture took place in Laboratories of the ProBioGen AG, Berlin.
Assays were conducted at the University of Applied Sciences Wildau, in the Laboratory for
Molecular Biotechnology and Functional Genomics.
NanoString experiments were conducted at the NanoString Technologies Inc. in Seattle (WA),
USA.
Acknowledgment of authors’ contributions (D)
Vaccine preparations from SF9 cells were generated by Stephan Hinderlich and Grit Sandig (Beuth
University of Applied Sciences, Berlin). Vaccine preparations from CHO cells, cell culture
experiment and freezing of cells were conducted by Christoph Giese and Annika Lubitz
(ProBioGen AG, Berlin). All sample extraction from frozen cells and qPCR assays were performed
by Lars Radke and Marcus Frohme (University of Applied Sciences Wildau).
Sample
Description (E)
Peripheral Blood Mononuclear Cells (PBMC) from whole blood or leukocyte concentrate of adult,
healthy donors were stimulated with vaccine preparations (RSV-F Protein, see M&M part). Cells
were harvested after 24 hours of stimulation. mRNA was extracted and reverse transcribed. cDNA
was used for gene expression analysis determination in qPCR and NanoString experiments.
Samples are assigned in accordance to their origin (PBMC or PBMC+mDC), their stimulation and
the concentration of the stimulant.
Volume/mass of sample processed (D)
3*106 cells (PBMC) and 1.5*106 cells (PBMC+mDC) were cultured per stimulation. Ca. 5*105 cells
were used for mRNA preparations from each stimulation type.
Microdissection or macrodissection (E)
PBMCs were derived from whole blood or leukocyte concentrate of adult, healthy donors.
Processing procedure (E)
For cell culture procedure see M&M part.
Total-RNA was isolated from stimulated cells. After control of the concentration, purity and quality
of the samples were reverse transcribed. mRNA was stored until used in qPCR or shipped for
analysis with an nCounter® Gene Expression assay (NanoString Technologies).
If frozen, how and how quickly? (E)
Cryoconservation of cells was performed immediately after collection and washing in cold medium
with 10% DMSO, 30% FCS and 60% RPMI 1640 final concentration. The vials were then
transferred into a pre-cooled (4°C) cryocontainer filled with isopropyl alcohol and placed in a −80°C
freezer overnight (cooling rate 1°C). Thereafter vials were stored in a nitrogen vapor phase.
Cultured Cells were frozen in RNAprotect Cell Reagent (Qiagen) at -80°C.
If fixed, with what and how quickly? (E)
Samples were not fixed.
Sample storage conditions and duration (especially for FFPE samples) (E)
Cryoconserved cells were stored in nitrogen vapor phase for up to 4 weeks.
Stimulated cells were stored at -156°C for less than a month.
Aliquots of extracted total-RNA samples were stored at -80°C for 4 weeks.
Nucleic acid extraction
Procedure and/or instrumentation (E)
Isolation of total-RNA from stimulated cells was carried out with a commercial kit and standard
laboratory equipment.
Name of kit and details of any modifications (E)
We used the High Pure RNA Isolation Kit (Roche) and performed the RNA-isolation as described
by the manufacturer.
Source of additional reagents used (D)
We did not use any additional reagents.
Details of DNase or RNase treatment (E)
We used DNase I which was included in the High Pure RNA Isolation Kit (Roche).
Contamination assessment (DNA or RNA) (E)
All samples were analyzed with a Nanodrop 1000. The ratio of the absorbance at 260 nm and 280
nm was used to assess the purity of the RNA.
Nucleic acid quantification (E)
We used the Nanodrop 1000 for mRNA quantification and analyzed 1.5 µl of sample.
Instrument and method (E)
Nanodrop 1000 (Nanodrop Instruments). Nucleic Acid method with RNA-40 as sample type.
Purity (A260/A280) (D)
We assessed the purity with the Nanodrop 1000 directly after total-RNA isolation:
sample name:
A260/A280:
PBMC NC
2.10
PBMC LPS
2.12
PBMC CHO 1.0
2.06
PBMC CHO 0.1
2.08
PBMC CHO 0.01
2.08
PBMC SF9 1.0
2.14
PBMC+mDC NC
2.18
PBMC+mDC LPS
2.14
PBMC+mDC CHO 1.0
2.09
PBMC+mDC CHO 0.1
2.11
PBMC+mDC CHO 0.01
2.07
PBMC+mDC SF9 1.0
2.10
All samples were analyzed a second time at the NanoString Technology Laboratory before
measuring them with the nCounter® Gene Expression assay:
sample name:
A260/A280:
PBMC NC
2.01
PBMC LPS
2.08
PBMC CHO 1.0
1.90
PBMC CHO 0.1
1.96
PBMC CHO 0.01
2.05
PBMC SF9 1.0
1.99
PBMC+mDC NC
1.98
PBMC+mDC LPS
2.06
PBMC+mDC CHO 1.0
2.10
PBMC+mDC CHO 0.1
2.07
PBMC+mDC CHO 0.01
2.05
PBMC+mDC SF9 1.0
2.08
Yield (D)
We assessed the yield with the Nanodrop 1000 directly after total-RNA isolation:
sample name:
Yield [ng/µl]
PBMC NC
50.76
PBMC LPS
37.65
PBMC CHO 1.0
48.60
PBMC CHO 0.1
42.41
PBMC CHO 0.01
42.70
PBMC SF9 1.0
50.30
PBMC+mDC NC
100.18
PBMC+mDC LPS
98.62
PBMC+mDC CHO 1.0
121.65
PBMC+mDC CHO 0.1
81.87
PBMC+mDC CHO 0.01
75.26
PBMC+mDC SF9 1.0
124.1
RNA integrity: method/instrument (E)
We assessed the RNA integrity with the Bioanalyzer 2100 (Agilent Technology) with a RNA pico
6000 assay as described by the manufacturer.
RIN/RQI or Cq of 3and 5 transcripts (E)
The following RIN values were determined:
sample name:
RIN
PBMC NC
9.50
PBMC LPS
9.50
PBMC CHO 1.0
8.60
PBMC CHO 0.1
8.80
PBMC CHO 0.01
9.00
PBMC SF9 1.0
9.50
PBMC+mDC NC
8.80
PBMC+mDC LPS
9.10
PBMC+mDC CHO 1.0
8.70
PBMC+mDC CHO 0.1
8.90
PBMC+mDC CHO 0.01
8.70
PBMC+mDC SF9 1.0
8.70
Electrophoresis traces (D)
Due to limited sample material we did not perform gel electrophoresis with the RNA extracts.
Instead we can supply the pseudo gel images from the Bioanalyzer runs:
Rev
erse
tran
scrip
tion
Com
plete
reac
tion
cond
ition
s (E)
Rev
erse
tran
scription was conducted with Maxima Reverse Transcriptase (Fermentas, now Thermo) and in
accordance to the protocol of the manufacturer.
In short:
Template, Primer and dNTPs were mixed and incubated at 65°C to melt any possible secondary
structure. To each sample 5.5 µl of mastermix were added, which contained 5x RT Buffer,
RiboLock RNase Inhibitor and Maxima Reverse Transcriptase. Samples were incubated at 25°C
and 50°C and the reaction was terminated by heating at 85°C for 5 min.
Amount of RNA and reaction volume (E)
From each sample 450 ng were reverse transcribed in a 20 µl reaction.
Priming oligonucleotide (if using GSP) and concentration (E)
We used random hexamer primer instead of Gene-specific Primers.
Reverse transcriptase and concentration (E)
1 µl (200 U) Maxima Reverse Transcriptase (Fermentas, now Thermo) were added within the
Mastermix to the total reaction volue of 20 µl.
Temperature and time (E)
- pre-incubation of total-RNA: 65°C for 5 min
- immediatly storage on ice (for addition of mastermix)
- reverse transcription at 25°C for 10 min followed by 50°C for 30 min
- termination of reaction at 85°C for 5 min
Manufacturer of reagents and catalogue numbers (D)
Primer: Thermo Scientific
dNTP: Thermo Fisher
5x RT Buffer Fermentas, now Thermo, #EP0742
Ribolock RNase Inhibitor: Fermentas, now Thermo, #EO0381
Maxima reverse transcriptase: Fermentas, now Thermo, #EP0742
Cq s with and without reverse transcription (D)
not examined.
Storage conditions of cDNA (D)
Alliquots of the cDNA were stored at -80°C.
qPCR target information
Gene symbol (E)
Sequence accession number (E) Amplicon length (E)
ALAS1
NM_000688
148 bp
B2M
NM_004048
146 bp
GAPDH
NM_002046
112 bp
HPRT1
NM_000194
178 bp
POLR2A
NM_000937
176 bp
POLR2F
NM_021974
157 bp
PPIA
NM_021130
115 bp
SDHA
NM_004168
144 bp
TBP
NM_003194
200 bp
CCL5
NM_002985
199 bp
IL12b
NM_002187
122 bp
TLR3
NM_003265
75 bp
In silico specificity screen (BLAST, and so on) (E)
See Appendix Blast.
Pseudogenes, retropseudogenes, or other homologs? (D)
There are 29 pseudogenes known for PPIA (PPIAP1-PPIAP29)
Location of each primer by exon or intron (if applicable) (E)
Gene
Exon Forward Primer
Exon Reverse Primer
ALAS1
10
11/12
B2M
2
4
GAPDH
1
2/3
HPRT1
7/8
9
POLR2A
19
20
POLR2F
2/3
4
PPIA
4
4/5
SDHA
5/6
6
TBP
7/8
8
CCL5
1
2/3
IL12b
5/6
6
TLR3
4
4
What splice variants are targeted? (E)
We used ensembl (http://www.ensembl.org) to get the sequences for our primer design. We used
protein coding transcripts only with highest coverage of exons from all listed transcripts.
qPCR oligonucleotides
Primer sequences (E)
Gene
Forward Primer
Reverse Primer
ALAS1
TGTGATGAACTAATGAGCAGAC
GTGACTAGCAGATTCTCAAGG
B2M
ACTGGTCTTTCTATCTCTTGTACT
CTTCAAACCTCCATGATGCT
GAPDH
CTCTGCTCCTCCTGTTCGAC
ACGACCAAATCCGTTGACTC
HPRT1
AGCCAGACTTTGTTGGATTT
ACTCAACTTGAACTCTCATCTTAG
POLR2A
CAGATGACCTTGAATACCTTCC
GCACAGAATATCCTTGGCTC
POLR2F
PPIA
AATGCCGAAGAGGGAAGGCCA
CAGGGGCACACATCGCAATC
TGAGAACTTCATCCTAAAGCATAC
CATCCAACCACTCAGTCTTG
TATATGGAAGGTCTCTGCGA
GTGTTCTTTGCTCTTATGCG
TATTAACAGGTGCTAAAGTCAGAG
AACTCAACATCCATCTTCTCAC
TACCATGAAGGTCTCCGC
GACAAAGACGACTGCTGG
GGACATCATCAAACCTGACC
AGGGAGAAGTAGGAATGTGG
TGGTTGGGCCACCTAGAAGTA
TCTCCATTCCTGGCCTGTG
SDHA
TBP
CCL5
IL12b
TLR3
RTPrimerDB identification number (D)
We used 2 Primer pairs from RTPrimerDB.
CCL5: ID 1650
TLR3: ID 3919
Probe sequences
not applicable (SYBR Green Assay)
Location and identity of any modifications (E)
We did not add any modification to the primers.
Manufacturer of oligonucleotides (D)
Thermo Scientific (http://www.thermohybaid.de/cgi-bin/start.app)
Purification method (D)
HPLC
qPCR protocol
Complete reaction conditions (E)
qPCR was performed on the LightCycler 480 (Roche) in 384 well plates. 5 pmol of equimolar
Primermix in 1 µl were placed on the bottom of each well and dried up by placing the plate for 30
min into a heating block. Samples were 1:50 diluted with double autoclaved DEPC treated water.
Mastermixes containing 5 µl of LightCycler® 480 SYBR Green I Master (Roche) and 5 µl of sample
for each reaction were mixed together and 10 µl were pipetted into the wells. qPCR experiments
were conducted in triplicates under the following conditions: 10 min of pre-incubation at 95°C
followed by 45 amplification cycles with 10 sec at 95°C, 60°C for 10 sec and 72°C for 15 sec
followed by melting curve acquisition from 65°C to 97°C with a ramp rate of 0.11°C per sec.
Reaction volume and amount of cDNA/DNA (E)
Reaction volume: 10µl with 9ng of cDNA.
Primer, (probe), Mg2, and dNTP concentrations (E)
5 pmol of equimolar Primermix in 1 µl were placed on the bottom of each well and dried up by
placing the plate for 30 min into a heating block.
The concentration of MgCl2 and dNTP within the LightCycler® 480 SYBR Green I Master is not
stated. The dNTP mix contains dUTP instead of dNTP.
Polymerase identity and concentration (E)
LightCycler® 480 SYBR Green I Master (Roche) contains „FastStart Taq DNA Polymerase“. A
concentration is not stated.
Buffer/kit identity and manufacturer (E)
LightCycler® 480 SYBR Green I Master (Roche)
Exact chemical composition of the buffer (D)
Not given in product sheet.
Additives (SYBR Green I, DMSO, and so forth) (E)
We used the LightCycler® 480 SYBR Green I Master (Roche) and did not add any other additives.
Manufacturer of plates/tubes and catalog number (D)
LightCycler®480Multiwell Plate 384 (Roche, #04729 749 001)
Tubes (A. Hartenstein Gesellschaft für Labor- und Medizintechnik mbH, Würzburg, Germany)
Complete thermocycling parameters (E)
pre-incubation 10 min at 95°C
45 amplification cycles:
10 sec at 95°C,
60°C for 10 sec
72°C for 15 sec
melting curve acquisition from 65°C to 97°C with a ramp rate of 0.11°C per sec.
Reaction setup (manual/robotic) (D)
Assay were pipetted manually.
Manufacturer of qPCR instrument (E)
LightCycler® 480 (Roche).
qPCR validation
Specificity (gel, sequence, melt, or digest) (E)
See Appendix “Melting Curves”.
For SYBR Green I, Cq of the NTC (E)
Means of NTC of all assayed genes are listed below. LightCycler 480 Software marks negative
amplification curves with a Cq value of 40. Mean Cqs that are lower than 40 are usually at least
about 10 Cq higher than the latest positive amplification.
Gene
Mean Cq of NTC
Gene
Mean Cq of NTC
ALAS1
40.0
PPIA
36.7
B2M
40.0
SDHA
40.0
GAPDH
40.0
TBP
40.0
HPRT1
40.0
CCL5
39.62
POLR2A
40.0
IL12b
34.10
POLR2F
32.0
TLR3
40.0
Calibration curves with slope and y intercept (E), PCR efficiency calculated from slope (E), CIs for
PCR efficiency or SE (D), r2 of calibration curve (E), Linear dynamic range (E)
Gene
Slope
Y intercept
Efficiency error r2
Linear Dynamic range
(in ng of cDNA)
ALAS1
-3.874
26.02
1.812
0.009 0.982 12 ng -0.75 ng
B2M
-3.634
16.60
1.884
0.040 0.922 12 ng -0.75 ng
GAPDH
-3.616
13.85
1.890
0.037 0.965 12 ng -0.75 ng
HPRT1
-3.380
23.46
1.976
0.013 0.971 12 ng -0.75 ng
POLR2A
-3.563
15.92
1.906
0.028 0.945 9 ng – 0.0145 ng
POLR2F
-3.461
21.59
1.945
0.009 0.982 12 ng -0.75 ng
PPIA
-4.128
20.82
1.747
0.020 0.960 12 ng -0.75 ng
SDHA
-3.561
18.30
1.909
0.024 0.953 12 ng -0.0469 ng
TBP
-3.584
26.22
1.818
0.013 0.975 12 ng -0.75 ng
CCL5
-3.896
19.15
1.806
0.033 0.935 12 ng -0.0469 ng
IL12b
-3.471
20.09
1.941
0.028 0.945 12 ng -0.1875 ng
TLR3
-3.425
27.05
1.959
0.031 0.939 12 ng -0.75 ng
Cq variation at LOD (E), Evidence for LOD (E)
LOD was not determined for all primer pairs, since reference genes are expressed constitutively
and biological expression range is within the determined linear range. Exemplary, for one gene
(POLR2A) the standard deviation for 0.0145 ng of input material (1:41250 fold dilution of 600 ng
cDNA) was 0.71.
Standard Deviation for target genes were 0.60 (CCL5), 0.03 (IL12b) and 0.50 (TLR3).
If multiplex, efficiency and LOD of each assay (E)
Assays were not performed in multiplex.
Data analysis
qPCR analysis program (source, version) (E)
LightCycler® 480 Software release 1.5.0 Version 1.5.0.39
GenEx 5.4.0.512, multid Analyses
Method of Cq determination (E)
2nd derivative maximum within LightCycler® 480 Software.
Outlier identification and disposition (E)
Test for Outlier in GenEx 5.4.0.512 (multid Analyses)
Results for NTCs (E)
LightcCycler 480 Software assigns a Cq value of 40 to negative amplification curves. Mean Cqs
that differ from 40 are usually at least about 10 Cq higher than the latest positive amplification.
Justification of number and choice of reference genes (E)
9 Reference Genes:
ALAS1, B2M, GAPDH, HPRT1, POLR2A, POLR2F, PPIA, SDHA, TBP
Choice of reference genes that were used for target gene normalization relied on comparison of
results from GeNorm, Normfinder and Bestkeeper.
Description of normalization method (E)
Normalization of target genes with geometric mean of PPIA, SDHA and TBP
or B2M, GAPDH and POLR2A.
Number and stage (reverse transcription or qPCR) of technical replicates (E)
3 technical replicates within the qPCR.
Repeatability (intraassay variation) (E)
The standard deviation of the technical replicates within one assay range from 0.0 to 0.48 (mean:
0.11; median: 0.06)
Statistical methods for results significance (E)
Little applicable in the context of the paper.
Software (source, version) (E)
LightCycler® 480 Software release 1.5.0 Version 1.5.0.39
GenEx 5.4.0.512, multid Analyses