Serum preparation, RNA isolation, and RT-qPCR assay
Venous blood samples (~5 ml) were collected from each patient and healthy volunteer. All the
samples were processed within 1 h. Serum separation was accomplished by centrifugation at
3,000 g for 10 min, followed by a 15 min high-speed centrifugation at 12,000 g to completely
remove cell debris. The supernatant serum was recovered and stored at -80 ℃ until analysis.
Total RNA was extracted from 100 μl of serum by phenol/chloroform purification and
centrifugation in isopropyl alcohol. Briefly, 100 μl of serum was mixed with 200 μl of phenol,
200 μl of chloroform, and 300 μl of RNase-free water. The sample was vortexed and
incubated at room temperature for 15 min. The mixture was centrifuged at 12,000 g for 10
min and the upper aqueous layer was collected. Subsequently, a 1/10 volume of 3 M sodium
acetate and a two-fold volume of isopropyl alcohol were added to the aqueous phase. The
total RNA was precipitated by incubation at -20 ℃ for 1 h. The RNA pellet was collected by
centrifugation at 16,000 g for 20 min, washed once with 75% ethanol, dried for 10 min at
room temperature, and then dissolved in 20 μl of RNase-free water.
TaqMan probe-based RT-qPCR assay was carried out using a commercial kit (Applied
Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, 2 μl
of total RNA was reverse transcribed to cDNA using AMV reverse transcriptase (TaKaRa,
Dalian, China) and the stem-loop RT primer (Applied Biosystems, Foster City, CA, USA).
Real-time PCR was performed using TaqMan miRNA probes on an Applied Biosystems 7300
Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The threshold
cycle (Cq) values were determined using the fixed threshold settings. All reactions, including
no-template controls, were performed in triplicate. To calculate the expression levels of target
miRNAs, a series of synthetic miRNA oligonucleotides with known concentrations were also
reverse transcribed and amplified to generate a standard curve. It was linear on a
semi-logarithmic plot in a range from 10 fM to 107 fM. The amount of each miRNA was then
calculated according to the standard curve. Because U6 and 5S rRNAs were degraded in the
serum samples, and there was no current consensus on housekeeping miRNAs for the
RT-qPCR analysis of serum miRNAs, the expression levels of miRNAs were directly
normalized to the serum volume in this study.
Supplementary Table 1 Differentially-expressed miRNAs of responders and
non-responders to chemotherapy in screening samples
order
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
name
mean fold
change
p-value
miR-20a
miR-21
miR-24
miR-25
miR-27b
miR-29a
miR-30a
miR-92a
miR-95
miR-99a
miR-100
miR-101
miR-107
miR-122
miR-125b
miR-130
miR-139-3p
miR-140-3p
miR-145
miR-150
miR-185
miR-191
3.13
4.24
2.67
2.08
1.07
1.1431785
1.4954582
4.14502
1.1179528
2.69
1.0234521
1.079218
1.094375
3.26
1.3597159
3.456
1.053803
1.042287
4.481299
1.746494
2.1
2.87
3.64E-07
2.28E-05
0.001255
8.18E-10
0.7567261
0.3623874
0.0731187
0.003084
0.5932393
2.61E-05
0.9108538
0.7229841
0.586468
0.004824
0.2975648
0.0054724
0.865472
0.7037122
0.001156
0.057758
0.007051
0.000372
significant
significant
significant
significant
non-significant
non-significant
non-significant
non-significant
non-significant
significant
non-significant
non-significant
non-significant
significant
non-significant
significant
non-significant
non-significant
significant
non-significant
significant
significant
23
24
25
26
27
28
29
30
31
32
33
34
35
miR-192
miR-197
miR-199a-3p
miR-210
miR-216
miR-320a
miR-361
miR-372
miR-411
miR-451
miR-483
miR-532-5p
miR-618
1.3246894 0.2644458
1.117953
0.593239
1.363585 0.2153148
1.3872444 0.3868377
3.732919 0.0044299
1.493328 0.0640379
1.5194114 0.3374292
4.0253
0.009875
1.796262
0.007805
1.7464941 0.0577575
5.632765 0.0009546
1.7962625 0.0078049
2.8435
0.00074587
non-significant
non-significant
non-significant
non-significant
significant
non-significant
non-significant
significant
non-significant
non-significant
significant
non-significant
significant