Experiment detail for qPCR in this study
Sample acquisition, handling, and preparation
All breast and liver tissue samples were collected by the University of Chicago Tissue
Core Facility. According to the record of the facility, all samples were frozen at -80℃
immediately after surgery and excluded the possibility of tumor by microscopical
examination. Before processing, the sample was embedded in O.C.T. compound (Ted
Pella, Redding, CA) and cut into 20μm slice in a cryostat.
RNA extraction and quality control
RNA was extracted by RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA)
according
to
manufacturer’s
recommendation
(http://www.qiagen.com/literature/render.aspx?id=170). The quantification and
A260/A280 is determined by NanoDrop 1000 (Thermo Scientific, Wilmington, DE). All
sample presented an A260/A280 ratio varying from 1.98-2.01, thus indicating that the
purity of our RNA sample was rather high. The RNA sample is not subjected to
DNase treatment. The integrity of RNA is examined by agrose gel electrophoresis.
Reverse transcription
cDNA for breast and liver samples was synthesized by using the High Capacity
Reverse Transcription Kit (Applied biosystems, Foster City, CA) according to
manufacturer’s recommendation. In detail, the reaction mix was setup as following:
10×RT buffer
25×dNTP Mix (100 mM)
10×RT Random Primers
MultiScrib Reverse Transcriptase
RNase Inhibitor
RNA
Nuclease-free H2O
Total volume
Volume (μL)
2.0
0.8
2.0
1.0
1.0
2μg
~
20
The following thermal condition was used: 25℃ for 10min, 37℃ for 120min, 85℃ for
5sec. The obtained cDNA was stored in -80℃ before using.
qPCR assay design
The primer for qPCR and related information is listed below. For each gene, the two
primers were located in different exons in order to avoid genomic DNA (gDNA)
contamination.
The
secondary
structure
is
determined
by
mfold
(http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi) and available on request. The
primer specificity was evaluated by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
and all primers were unique in human genome. All PCR products were also sequenced
to confirm the primer specificity. All qPCR assays presented a single and sharp peak
in melt curve.
Gene
referencea
Forward primer (5’-3’)
location
Reverse primer (5’-3’)
location
UGT2B4
NM_021139
AACCTGCCAAACCCCTACCG
895-914
TCCACTTGTACAGCCGAGTATTGAGT
1091-1116
UGT2B7 b
NM_001074
TGCTTTACTTTGACTTTTGGTTCG
696-719
CCAGGAGTTTCGAATAAGCCATAC
809-832
UGT2B10
NM_001075
ACAGCTTCAGTCCTGGCTACT
537-557
AAGCCATATGTCAGCTTTCCT
773-793
UGT2B15
NM_001076
TGAAGTCAGTCATTAATGACCCTG
1327-1350
CTCGATCCAGGGGCTTCAT
1401-1419
UGT2B17
NM_001077
TATTTTGACTTTTGGTTTCAAGC
701-723
CTCTTCCATTTCCTTAGGCAA
908-928
ß-actin c
NG_007992
ACGTGGACATCCGCAAAGAC
2472-2491
CAAGAAAGGGTGTAACGCAACTA
2869-2891
K03432
GTAACCCGTTGAACCCCATT
1718-1737
CCATCCAATCGGTAGTAGCG
1849-1868
18S
d
aGenbank
ID provided.
reference 1.
cfrom reference 2.
d
from reference 3.
bfrom
qPCR setup
The qPCR was performed with power SYBR green (AB#4367659, Applied
Biosystems) and the exact chemical composition is available in vendor website
(https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&cat
ID=602649). The reaction was setup as following:
Power SYBR green master mix
Forward primer (10μM)
Reverse primer (10μM)
water
cDNA
Total volume
10μL
1μL
1μL
3μL
5μL (10 ng)
20μL
All qPCR reaction is performed in optical plate (AB#4346906, Applied Biosystems)
and sealed with optical adhesive film (AB#4311971, Applied Biosystems). The
annealing and extension temperature was fixed at 60℃ according to manufacturer’s
recommendation and thermal condition was:
95℃ for 10min; 95℃ for 15sec and 60℃ for 1min, 40cycles.
All qPCR were performed in triplicate on a StepOne Plus Real time PCR System
(Applied Biosystems).
The gDNA contamination was evaluated by qPCR as described in our recent report 4.
The result indicated that gDNA contamination is rather low (<5% for most cases).
Moreover, since our qPCR primer is in different exons, the gDNA contamination was
not likely to influence qPCR result.
qPCR assay performance
A cDNA mix was 1:5 serial diluted as calibration curve. In each reaction, 100, 20, 4,
0.8, 0.16, 0.032ng cDNA was used and the linear dynamic range covered all sites (all
r2>0.99). All unknown samples were within the linear range. The slope and intercept
for calibration curve were provided by StepOne software (Applied Biosystems). The
PCR efficiency was calculated with the formula 10-1/slope-1 by StepOne software and
listed in below table.
Gene
UGT2B4
UGT2B7
UGT2B10
UGT2B15
UGT2B17
actin
18S
aMean
Efficiency (mean±standard deviation)a
98.5±4.4%
95.2±3.6%
100.4±2.6%
111.9±3.2%
99.2±6.1%
95.6±1.9%
102.2±2.4%
and standard deviation calculated from the replicates.
In above table, an efficiency value beyond 100% was observed for multiple genes.
Although it is difficult to be interpreted by PCR theory, it is not uncommon in qPCR
practice 5-7.
In each plate, a non-reverse transcribed RNA sample and H2O sample were included
and both of them were not amplified.
Data analysis
The copy number for each sample was calculated by StepOne software and outputted
into spreadsheet. The mean and standard deviation (SD) for each sample was
calculated by SPSS 15.0 (SPSS Inc., Chicago, IL). Coefficient of variation (CV) is
determined by the formula SD/mean. All samples presented a CV<20%, thus
indicating that our result was highly repeatable.
In a preliminary experiment, the expression of ß-actin, 18S, GAPDH (primer pair
5’-GAAGGTGAAGGTCGGAGTC-3’ and 5’-GAAGATGGTGATGGGATTTC-3’) 8,
and
U6
RNA
(primer
pair
5’-CTCGCTTCGGCAGCACA-3’
and
5’-AACGCTTCACGAATTTGCGT-3’) 9 was evaluated in both breast and liver
samples. β-actin and 18S were chosen as reference gene for breast and liver,
respectively, due to their conserved expression in these two tissues (result not shown).
The final mRNA level is expressed as the ratio between target gene of interest and
ß-actin or 18S. After log-transform, all genes were in normal distribution and used for
further analysis.
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