Supplementary Material to Rouvinen-Watt et al., page 1
Supplementary Material to
Evidence of endoplasmic reticulum stress and liver inflammation in the American mink
Neovison vison with benign hepatic steatosis by Rouvinen-Watt et al.
CONTENT
Doc S1 of Supplementary Material…………………………………………….……………….1
Author contributions ..................................................................................................................... 1
RNA extraction ............................................................................................................................. 1
Reverse transcription .................................................................................................................... 1
qPCR protocol .............................................................................................................................. 2
Validation ..................................................................................................................................... 2
Data analysis ................................................................................................................................. 2
MIQE checklist ............................................................................Error! Bookmark not defined.
Table S1 of Supplementary Material…………………………………………….……………..7
Table S2 of Supplementary Material…………………………………………….……………..7
Table S3 of Supplementary Material…………………………………………….……………..8
References to Supplementary Material…………………………………………….…………..8
Author contributions
K. Rouvinen-Watt, P. Nieminen, and A.-M. Mustonen planned this research and KRW oversaw
all experimental procedures. C. Pal, T. Martin, L. Harris, D. Kryzskaya, and V. Kärjä carried out
the laboratory analyses. Data analysis was performed by KRW, T. Astatkie, CP, TM, and PN. R.
Tammi and M. Tammi contributed to the interpretations of the data. Manuscript writing was led
by KRW, with contributions from all co-authors.
RNA extraction
Mink livers were obtained (by macrodissection) within 15–25 min from time of death. Samples
were immediately snap frozen in liquid nitrogen and kept at –85°C until RNA extraction, six
months later. Total RNA content was extracted from approximately 30 mg tissue, with
homogenization using the Kimble® Kontes Pellet Pestle and QIAShredder homogenizers. RNA
was treated with RNase free DNase I (Qiagen, Toronto, ON, Canada) using the manufacturer’s
on-column treatment protocol. There was an average yield of 2.28 ± 0.06 µg RNA/mg tissue.
Sample purity was assessed by an A260:A280 ratio of 1.8–2.1.
Reverse transcription
Total RNA (1.0 µg) was reverse transcribed with 2.5 U/µL MultiScribe™ Reverse Transcriptase
in a reaction volume of 20 µL at 37°C for 120 min, using the High Capacity cDNA Reverse
Transcription Kit with RNase inhibitor, catalog# 4374966 (Applied Biosystems, Carlsbad, CA,
USA). 0.1X and 0.01X dilutions of cDNA were prepared in 10 mM Tris-Cl, pH=7.6, and stored
Supplementary Material to Rouvinen-Watt et al., page 2
at –30°C. GAPDH qPCR run on a set of no reverse transcription controls showed no genomic
contamination in 42 samples, minor contamination (Cq=31.2) in one sample, and trace
contamination in the remaining 17 samples (Cq>35).
qPCR protocol
Primers, purified by reverse-phase cartridge separation, were purchased from Sigma (SigmaAldrich Canada Co, Oakville, ON, Canada). Assays were run in a reaction volume of 10 µL,
using 1 µL of 0.1X diluted cDNA, using 384-well white plates, catalog #04729749001 (Roche
Diagnotics Canada, Laval, QC, Canada). Each sample was run with three technical replicates at
the qPCR stage, and each treatment group included five technical replicates. All assays except
GRP78 were run with 1X GoTaq® Flexi buffer, 2.5 mM MgCl2, 0.2 mM dNTP (each), 0.8 U
GoTaq® HotStart Polymerase (Promega, Fitchburg, WI, USA), and 1X EvaGreen® (Biotium
Inc, Hayward, CA, USA). The GRP78 assay was run using 1X SsoFastTM EvaGreen® mastermix
(Bio-Rad Laboratories Inc., Hercules, CA, USA), 500 nM primers, with a hot start at 95°C for 2
min followed by 35 cycles of denaturation at 95°C for 5 s, combined annealing and extension at
58°C for 20 s.
Validation
The specificity of each qPCR assay was confirmed by verification of the size of the amplicon on
agarose gel electrophoresis (gel photographs available on request), and the presence of a single
peak in a melt curve. No amplification was seen in the no template controls (n=6). The limit of
detection for each assay was at or below the lower limit of the linear dynamic range as reported
in Table S3, as shown by a linear standard curve to this point. To test for inhibition, qPCR results
of standards spiked with pooled cDNA (0.1X dilutions) were compared with those of pooled
cDNA (0.1X dilution) alone, and standards run separately. No inhibition was seen.
Data analysis
Lightcycler® 480 Software, release 1.5.0.39 (Roche, Indianapolis, IN, USA) was used to obtain
quantification cycles (Cq) results for each sample, employing the “second derivative maximum”
method. Each qPCR run included a standard curve (a 5-fold dilution series from a known amount
of target cDNA, created from synthetic copy RNA), which was used to calculate the expression
of the target gene in each sample. Each run was examined for anomalous amplification curves,
and all were within acceptable norms, so that no outliers were removed. To normalize each
sample, the geometric mean of the replicate concentrations of a target gene was divided by the
geometric mean of the replicate concentrations of the reference gene.
Supplementary Material to Rouvinen-Watt et al., page 3
MIQE Checklist according to Bustin et al. (2009)
ITEM TO CHECK
IMPORTANCE
CHECKLIST
WHERE IN THE MANUSCRIPT
Definition of experimental and control groups
E
Yes
Methods and materials: Animal experiment
Number within each group
E
Yes
Methods and materials: Animal experiment
Assay carried out by core lab or investigator's lab?
D
Yes
All experiments carried out in investigator’s lab.
Acknowledgement of authors' contributions
D
Yes
S1: Author contributions
E
Yes
Methods and materials: Quantitative real-time
polymerase chain reaction
Volume/mass of sample processed
D
Yes
S1: RNA extraction
Microdissection or macrodissection
E
Yes
S1: RNA extraction
E
Yes
S1: RNA extraction
If frozen - how and how quickly?
E
Yes
S1: RNA extraction
If fixed - with what, how quickly?
E
Yes
S1: RNA extraction
E
Yes
S1: RNA extraction
EXPERIMENTAL DESIGN
SAMPLE
Description
Processing procedure
Sample storage conditions and duration (especially for FFPE samples)
NUCLEIC ACID EXTRACTION
Procedure and/or instrumentation
Methods and materials: Quantitative real-time
polymerase chain reaction
Methods and materials: Quantitative real-time
polymerase chain reaction
E
Yes
Name of kit and details of any modifications
E
Yes
Source of additional reagents used
D
n/a
Details of DNase or RNase treatment
E
Yes
S1: Validation
Contamination assessment (DNA or RNA)
E
Yes
Nucleic acid quantification
E
Yes
Instrument and method
E
Yes
S1: Reverse transcription
Methods and materials: Quantitative real-time
polymerase chain reaction
Methods and materials: Quantitative real-time
polymerase chain reaction
Purity (A260/A280)
D
Yes
S1: RNA extraction
Yield
D
Yes
S1: RNA extraction
E
Yes
Methods and materials: Quantitative real-time
RNA integrity method/instrument
Supplementary Material to Rouvinen-Watt et al., page 4
polymerase chain reaction
RIN/RQI or Cq of 3' and 5' transcripts
E
ND
Electrophoresis traces
D
Yes
Available on request
E
Yes
S1: RNA extraction
E
Yes
Methods and materials: Quantitative real-time
polymerase chain reaction and S1
Amount of RNA and reaction volume
E
Yes
S1: Reverse transcription
Priming oligonucleotide (if using GSP) and concentration
E
N/A
Inhibition testing (Cq dilutions, spike or other)
REVERSE TRANSCRIPTION
Complete reaction conditions
E
Yes
Methods and materials: Quantitative real-time
polymerase chain reaction and S1: Reverse
transcription
Temperature and time
E
Yes
S1: Reverse transcription
Manufacturer of reagents and catalogue numbers
D
Yes
S1: Reverse transcription
Cqs with and without RT
D*
Yes
S1: Reverse transcription
Storage conditions of cDNA
D
Yes
S1: Reverse transcription
If multiplex, efficiency and LOD of each assay.
E
N/A
Sequence accession number
E
Yes
Table 1
Location of amplicon
D
Yes
S1: Table S1
Amplicon length
E
Yes
Table 1
In silico specificity screen (BLAST, etc)
E
Yes
Primer BLAST run for all targets (Ye et al. 2012)
Pseudogenes, retropseudogenes or other homologs?
D
No
Information unavailable for mink.
D
No
D
Yes
All amplicons analyzed using Mfold (Zuker 2003)
E
Yes
S1: Table S1
E
No
Splice variation information unavailable for mink.
Primer sequences
E
Yes
Table 1
RTPrimerDB Identification Number
D
N/A
Reverse transcriptase and concentration
qPCR TARGET INFORMATION
Sequence alignment
Secondary structure analysis of amplicon
Location of each primer by exon or intron (if applicable)
What splice variants are targeted?
qPCR OLIGONUCLEOTIDES
Supplementary Material to Rouvinen-Watt et al., page 5
Probe sequences
D**
N/A
Location and identity of any modifications
E
Yes
None
Manufacturer of oligonucleotides
D
Yes
S1: qPCR protocol
Purification method
D
Yes
S1: qPCR protocol
E
Yes
Methods and materials: Quantitative real-time
polymerase chain reaction
Reaction volume and amount of cDNA/DNA
E
Yes
S1: qPCR Protocol
Primer, (probe), Mg++ and dNTP concentrations
E
Yes
S1: Table S2 and qPCR protocol
Polymerase identity and concentration
E
Yes
S1: qPCR protocol
Buffer/kit identity and manufacturer
E
Yes
S1: qPCR protocol
Exact chemical constitution of the buffer
D
No
Not available from manufacturer.
Additives (SYBR Green I, DMSO, etc.)
E
Yes
Manufacturer of plates/tubes and catalog number
D
Yes
Complete thermocycling parameters
E
Yes
Reaction setup (manual/robotic)
D
Yes
Manufacturer of qPCR instrument
E
Yes
S1: qPCR protocol
Methods and materials: Quantitative real-time
polymerase chain reaction and S1: qPCR protocol
Methods and materials: Quantitative real-time
polymerase chain reaction, S1: Table S2, and S1:
qPCR protocol
Methods and materials: Quantitative Real-Time
Polymerase Chain Reaction
Methods and materials: Quantitative real-time
polymerase chain reaction
Evidence of optimization (from gradients)
D
Yes
Available on request
Specificity (gel, sequence, melt, or digest)
E
Yes
S1: Validation
For SYBR Green I, Cq of the NTC
E
Yes
S1: Validation
Standard curves with slope and y-intercept
E
Yes
S1: Table S3
PCR efficiency calculated from slope
E
Yes
S1: Table S3
Confidence interval for PCR efficiency or standard error
D
No
E
Yes
S1: Table S3
E
Yes
S1: Table S3
qPCR PROTOCOL
Complete reaction conditions
qPCR VALIDATION
2
r of standard curve
Linear dynamic range
Supplementary Material to Rouvinen-Watt et al., page 6
Cq variation at lower limit
E
Yes
S1: Table S3
Confidence intervals throughout range
D
No
Evidence for limit of detection
E
Yes
If multiplex, efficiency and LOD of each assay.
E
N/A
E
Yes
S1: Data analysis
Cq method determination
E
Yes
S1: Data analysis
Outlier identification and disposition
E
Yes
S1: Data analysis
Results of NTCs
E
Yes
Justification of number and choice of reference genes
E
Yes
Description of normalisation method
E
Yes
Number and concordance of biological replicates
D
Yes
S1: Validation
Methods and materials: Quantitative real-time
polymerase chain reaction
Methods and materials: Quantitative real-time
polymerase chain reaction and S1: Data analysis
Methods and materials: Animal experiment and
S1: qPCR protocol
Number and stage (RT or qPCR) of technical replicates
E
Yes
S1: qPCR protocol
Repeatability (intra-assay variation)
E
Yes
S1: Table S3
Reproducibility (inter-assay variation, %CV)
D
No
Power analysis
D
No
Statistical methods for result significance
E
Yes
Methods and materials: Statistical analysis
Software (source, version)
E
Yes
Methods and materials: Statistical analysis
Cq or raw data submission using RDML
D
No
S1: Validation
DATA ANALYSIS
qPCR analysis program (source, version)
Supplementary Material to Rouvinen-Watt et al., page 7
Table S1 Target information for qPCR primer pairs.
Sequence
Location of
Amplicon
Location of forward
Location of reverse
accession number
amplicon
length
primer (human exon)
primer (human exon)
GAPDH
AF076283
36-116
84
7
7
GRP78
HQ003898
501-611
111
8
8
LPL
AJ223493
1813-1908
96
10
10
MCP-1
HQ163895
149-266
118
2
3
TNF-a
GU327784
81-205
125
3
4
Target gene
Table S2 Thermocycling parameters for qPCR assays.
Concentration
primers (nM)
400
Number of
cycles
40
Annealing
temperature (°C)
60
Denaturation
time (s)
10
Annealing time
(s)
20
Extension time
(s)
30
LPL
400
45
60
10
20
30
MCP-1
400
45
60
30
30
30
TNF-a
400
45
62
30
30
30
Target gene
GAPDH
Supplementary Material to Rouvinen-Watt et al., page 8
Table S3 qPCR validation for qPCR assays.
Target
Standard curve
Linear dynamic
Cq variation at
Repeatability (intra-assay
range (copies)
lower limit
variation) (CV of Cq, %)
103-108
0.07
0.10
Y-intercept
Efficiency (%)
r2
41.69
93.8
0.9999
–3.542
42.28
91.6
0.9990
103-108
0.00
0.25
LPL
–3.513
43.29
92.6
0.9988
102-108
0.82
0.19
MCP-1
–3.465
47.41
94.4
0.9968
103-108
0.43
0.46
TNF-a
–3.455
40.05
94.7
0.9968
25-108
0.06
1.11
gene
Slope
GAPDH
–3.481
GRP78
References to Supplementary Material
Bustin SA, Benes V, Garson JA et al (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time
PCR Experiments. Clin Chem 55:611–622
Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction.
BMC Bioinformatics 13:134
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415