SUPPLEMENTARY MATERIALS AND METHODS
Sample Processing
Whole blood was collected from all children according to RCH standard operating
procedures in BD Vacutainer Plus plastic serum tubes (Becton, Dickinson & Co, USA),
containing clot activator and gel for serum separation. Serum was isolated by centrifugation
at 913 g for 10 min at room temperature in a GS-6 centrifuge (Beckman Coulter, USA),
aliquoted into 1 mL Eppendorf tubes and stored at -80 ˚C, in the absence of freeze-thaw
cycles, until analysis.
RNA Extraction
Serum was thawed on ice and spun for 5 min at 3000 g in an Eppendorf 5415D benchtop
centrifuge (Eppendorf, Germany) to pellet any debris. Prior to RNA extraction,
spectrophotometry was carried out on serum samples to test for hemolysis by measuring
the absorbance of free hemoglobin at 414 nm. Samples with OD414 greater than 0.2 were
excluded from the study (1), due to the potential of cellular miRNAs to confound the results.
For the preliminary PCR array, RNA was extracted from 200 µL serum using the miRNeasy
Mini Kit (Qiagen, Germany) as per the manufacturer’s instructions. For the validation RTqPCR experiments, RNA was extracted from 200 µL serum using the miRCURY RNA Isolation
Kit for Biofluids (Exiqon, Denmark), according to the manufacturer’s protocol. All purified
RNA was eluted in 50 µL RNase-free water, split into single-use aliquots and stored at -80 ˚C.
RNA Quality
Due to the low concentration of RNA in serum it was not possible to measure the RNA
concentration and purity using spectrophotometry; our samples were at or below the limit
of detection of the Nanodrop (Thermo Scientific, USA; data not shown). We considered
using the Bioanalyzer (Agilent Technologies, USA) to determine RNA integrity, however, this
method relies on the ribosomal RNA fraction, which was excluded by the commercial kits
used during the RNA isolation step. Although a small RNA chip is available for quantifying
RNA species <200 nucleotides, it is recommended to measure miRNA as a proportion of the
total RNA, rather than as a single fraction (2). Because of these factors we followed the
advice of Exiqon and others (3-5) and used a fixed volume of eluted RNA, rather than a fixed
RNA mass, in our reverse transcription reactions.
Reverse Transcription
For the preliminary PCR array, RNA was reverse transcribed using the miScript II RT kit
(Qiagen). In 1 mL PCR tubes, 5 µL RNA was added to 15 µL master mix, comprised of 4 µL 5x
HiSpec Buffer, 2 µL 10x Nucleics Mix, 2 µL RT Mix and 7 µL nuclease-free water. Tubes were
incubated at 37 ˚C for 1 h in a water bath. cDNA was diluted 1:11 in nuclease-free water and
stored at -80 ˚C. For the validation RT-qPCR experiments, RNA was reverse transcribed for
each sample in triplicate using the miRCURY LNA Universal cDNA Synthesis Kit II (Exiqon).
Four µL RNA was added to 200 µL PCR tubes containing 16 µL master mix, comprised of 4 µL
5x Reaction Buffer, 2 µL enzyme mix, 9 µL nuclease-free water and 1 µL UniSp6 synthetic
spike-in control, used to monitor the efficiency of the RT step. Reactions were incubated in a
PTC-100 thermal cycler (MJ Research, USA) at 60 ˚C for 60 min, followed by 95 ˚C for 5 min
and holding at 4 ˚C. cDNA was split into single use aliquots and stored at -80 ˚C. Reactions
containing nuclease-free water in place of enzyme and/or RNA template served as negative
controls; these controls were negative in all PCR experiments.
RT-qPCR
We first conducted a PCR array (Human Serum & Plasma miRNA PCR Array; Qiagen, Cat. No.
MIHS-106Z) to profile the expression of 84 miRNAs detectable in serum in order to identify
candidate miRNAs for subsequent validation. A master mix was prepared for each cDNA
sample as follows: 100 µL diluted cDNA, 1375 µL 2x QuantiTect SYBR Green PCR Master Mix,
275 µL 10x miScript Universal Primer and 1 mL nuclease-free water. Reactions were set up
in 96 well plates in a final volume of 10 µL for each miRNA in the array as per the
manufacturer’s instructions. PCR amplification was carried out in the Roche Light Cycler
LC480 (Roche, Switzerland). Samples were initially denatured at 95 ˚C for 15 min, followed
by 40 cycles of: denaturation at 94 ˚C for 15 sec, annealing at 55 ˚C for 30 sec, extension at
70 ˚C for 30 sec. Fluorescence data were collected after the extension step. Validation RTqPCR experiments on candidate miRNAs were set up as follows: 8 µL cDNA (diluted 1:40 in
nuclease-free water) was added to Qiagen 100 µL PCR strip tubes containing 12 µL master
mix, comprised of 10 µL ExiLENT SYBR Green master mix (Exiqon) and 2 µL miRCURY LNA
PCR primer mix (Exiqon; see Supplementary Table 1). PCR amplification was carried out in
the Corbett Rotor-Gene 3000 or 6000 machine (Qiagen), with cycling parameters as follows:
initial denaturation of 95 ˚C for 10 min, followed by 40 cycles of: 60 ˚C for 1 min and 95 ˚C
for 10 sec. Fluorescence data were collected after the 60 ˚C step. Melt curve analysis was
carried out to confirm the production of a single amplicon by heating tubes from 55 ˚C to 90
˚C, with fluorescence measurements obtained in one degree increments. A set of standards
(run in triplicate) was included in each run, comprising five-fold serial dilutions made from
aliquots of cDNA prepared from a pool of all RNA samples. A set of identical inter-run
calibrators (IRC) was also included in at least duplicate in each run, consisting of cDNA
prepared from a pool of all RNA samples and diluted 1:33 in nuclease-free water
immediately prior to PCR.
Data Analysis
Quantification cycle (Cq) values were determined using the Roche LightCycler 480 software
v1.5 default threshold settings (for the PCR array) or Rotor-Gene 6000 series software v1.7
default threshold settings based on each assay’s standard curve (for the validation RTqPCR). PCR efficiencies were determined for each run and were acceptable when slopes
were in the range of -3.2 and -3.5, with R2 values over 0.98 (6). Raw Cq values were input
into qbase+ v2.5 software (Biogazelle, Belgium) (7) in order to calculate normalized relative
quantities of each miRNA in the Qiagen PCR array and thus to identify candidate miRNAs
that would be validated in the subsequent RT-qPCR experiments. The qbase+ software
contains the geNorm tool (8), which was used to determine the most stable reference
miRNAs out of the detectable miRNAs in the array as well as the optimal number of
reference miRNAs for accurate data normalization. Raw Cq values of the candidate miRNAs
were also input into qbase+, with data normalized to the most stable reference miRNAs
identified in the array (miR-93 and miR-19b). qbase+ automatically scaled data based on
differences in IRC Cq values, and we also identified and excluded samples with a reduced RT
efficiency by taking into account differences in Cq values for the synthetic spike-in control,
UniSp6, between RT triplicates (9). The normalized relative quantities calculated by qbase+
were then subjected to statistical analysis as described.
SUPPLEMENTARY REFERENCES
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Haemolysis during sample preparation alters microRNA content of plasma. PLoS ONE
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for RT-qPCR analysis. Methods 2010;50:237-43.
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quantification framework and software for management and automated analysis of real-time
quantitative PCR data. Genome Biol 2007;8(R19).
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Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple
internal control genes. Genome Biol 2002;3(7).
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SUPPLEMENTARY FIGURE LEGEND
Supplementary Figure 1: Results of reference gene validation study using geNorm. (A) shows
calculated M values for the 10 most stable miRNAs detected in the PCR array, ranked in
order of stability. The most stable miRNAs are seen at the right of the chart. In (B), pairwise
variation analysis determined that two reference genes (i.e. miR-93 and miR-19b) were
optimal for accurate normalization, indicated at the V2/3 step of the chart, where the V
threshold of below 0.15 is met.