Reference Gene Stability in Peripheral Blood Mononuclear Cells Determined by Qpcr And

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
PBMC LPS
PBMC CHO 1.0
PBMC CHO 0.1
PBMC CHO 0.01
PBMC SF9 1.0
PBMC+mDC NC
PBMC+mDC LPS
PBMC+mDC CHO 1.0
PBMC+mDC CHO 0.1
PBMC+mDC CHO 0.01
PBMC+mDC SF9 1.0 / 2.10
2.12
2.06
2.08
2.08
2.14
2.18
2.14
2.09
2.11
2.07
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
PBMC LPS
PBMC CHO 1.0
PBMC CHO 0.1
PBMC CHO 0.01
PBMC SF9 1.0
PBMC+mDC NC
PBMC+mDC LPS
PBMC+mDC CHO 1.0
PBMC+mDC CHO 0.1
PBMC+mDC CHO 0.01
PBMC+mDC SF9 1.0 / 2.01
2.08
1.90
1.96
2.05
1.99
1.98
2.06
2.10
2.07
2.05
2.08

Yield (D)

We assessed the yield with the Nanodrop 1000 directly after total-RNA isolation:

sample name: / Yield [ng/µl]
PBMC NC
PBMC LPS
PBMC CHO 1.0
PBMC CHO 0.1
PBMC CHO 0.01
PBMC SF9 1.0
PBMC+mDC NC
PBMC+mDC LPS
PBMC+mDC CHO 1.0
PBMC+mDC CHO 0.1
PBMC+mDC CHO 0.01
PBMC+mDC SF9 1.0 / 50.76
37.65
48.60
42.41
42.70
50.30
100.18
98.62
121.65
81.87
75.26
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
PBMC LPS
PBMC CHO 1.0
PBMC CHO 0.1
PBMC CHO 0.01
PBMC SF9 1.0
PBMC+mDC NC
PBMC+mDC LPS
PBMC+mDC CHO 1.0
PBMC+mDC CHO 0.1
PBMC+mDC CHO 0.01
PBMC+mDC SF9 1.0 / 9.50
9.50
8.60
8.80
9.00
9.50
8.80
9.10
8.70
8.90
8.70
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:

Reverse transcription

Complete reaction conditions (E)

Reverse transcription 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
B2M
GAPDH
HPRT1
POLR2A
POLR2F
PPIA
SDHA
TBP
CCL5
IL12b
TLR3 / NM_000688
NM_004048
NM_002046
NM_000194
NM_000937
NM_021974
NM_021130
NM_004168
NM_003194
NM_002985
NM_002187
NM_003265 / 148 bp
146 bp
112 bp
178 bp
176 bp
157 bp
115 bp
144 bp
200 bp
199 bp
122 bp
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
B2M
GAPDH
HPRT1
POLR2A
POLR2F
PPIA
SDHA
TBP
CCL5
IL12b
TLR3 / 10
2
1
7/8
19
2/3
4
5/6
7/8
1
5/6
4 / 11/12
4
2/3
9
20
4
4/5
6
8
2/3
6
4

What splice variants are targeted? (E)

We used ensembl ( 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
B2M
GAPDH
HPRT1
POLR2A
POLR2F
PPIA
SDHA
TBP
CCL5
IL12b
TLR3 / TGTGATGAACTAATGAGCAGAC
ACTGGTCTTTCTATCTCTTGTACT
CTCTGCTCCTCCTGTTCGAC
AGCCAGACTTTGTTGGATTT
CAGATGACCTTGAATACCTTCC
AATGCCGAAGAGGGAAGGCCA
TGAGAACTTCATCCTAAAGCATAC
TATATGGAAGGTCTCTGCGA
TATTAACAGGTGCTAAAGTCAGAG
TACCATGAAGGTCTCCGC
GGACATCATCAAACCTGACC
TGGTTGGGCCACCTAGAAGTA / GTGACTAGCAGATTCTCAAGG
CTTCAAACCTCCATGATGCT
ACGACCAAATCCGTTGACTC
ACTCAACTTGAACTCTCATCTTAG
GCACAGAATATCCTTGGCTC
CAGGGGCACACATCGCAATC
CATCCAACCACTCAGTCTTG
GTGTTCTTTGCTCTTATGCG
AACTCAACATCCATCTTCTCAC
GACAAAGACGACTGCTGG
AGGGAGAAGTAGGAATGTGG
TCTCCATTCCTGGCCTGTG

RTPrimerDB identification number (D)

We used 2 Primer pairs from RTPrimerDB.

CCL5: ID 1650TLR3: 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 (

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
B2M
GAPDH
HPRT1
POLR2A
POLR2F / 40.0
40.0
40.0
40.0
40.0
32.0 / PPIA
SDHA
TBP
CCL5
IL12b
TLR3 / 36.7
40.0
40.0
39.62
34.10
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
B2M
GAPDH
HPRT1
POLR2A
POLR2F
PPIA
SDHA
TBP
CCL5
IL12b
TLR3 / -3.874
-3.634
-3.616
-3.380
-3.563
-3.461
-4.128
-3.561
-3.584
-3.896
-3.471
-3.425 / 26.02
16.60
13.85
23.46
15.92
21.59
20.82
18.30
26.22
19.15
20.09
27.05 / 1.812
1.884
1.890
1.976
1.906
1.945
1.747
1.909
1.818
1.806
1.941
1.959 / 0.009
0.040
0.037
0.013
0.028
0.009
0.020
0.024
0.013
0.033
0.028
0.031 / 0.982
0.922
0.965
0.971
0.945
0.982
0.960
0.953
0.975
0.935
0.945
0.939 / 12 ng -0.75 ng
12 ng -0.75 ng
12 ng -0.75 ng
12 ng -0.75 ng
9 ng – 0.0145 ng
12 ng -0.75 ng
12 ng -0.75 ng
12 ng -0.0469 ng
12 ng -0.75 ng
12 ng -0.0469 ng
12 ng -0.1875 ng
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 were0.60 (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