Clinical samples
This study was approved by the ethics committee of the Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences. Surgical material was obtained in compliance with the legislation of the Russian Federation, and written informed consent was provided by all the patients. 208 tissue samples from thyroid nodules were surgically resected from patients undergoing thyroidectomy (28 men and 180 women, median age 54 and 56 years, respectively). The samples, collected between 2011 and 2013, represent a workflow of a house surgeon during about 2.5 years. Samples had not been pre-selected intentionally, so the proportion of lesion types reflects the real distribution of thyroidectomy cases in the setting of study. Nevertheless, due to various reasons, mostly technical, 12 patients were excluded from analysis, including one case of anaplastic thyroid carcinoma and one case of poorly differentiated squamous cell carcinoma. Adjacent non-tumor thyroid tissue served as a control. Special care was taken to ensure that no adjacent normal tissue was present in the tumor sample, and vice versa. Sample collection and histology analysis were controlled by a qualified oncologist (Oncology department VI, Novosibirsk Municipal Clinical Hospital #1). Upon resection, tissue samples were immediately placed in EverFresh RNA solution (SILEX, Russia) and stored at +4-8 °C for up to a week until processed. Demographic and clinical characteristics of the samples are shown in [Additional file 1].
Histopathology analysis
Tissue samples were processed according to the standard protocol, i.e., tumor pieces and regional lymph nodes were fixed in 10 % neutral buffered formalin, dehydrated in a graded series of alcohols, cleared in xylol and embedded in paraffin. 5 μm thick paraffin sections were stained with hematoxylin and eosin. This was followed by light microscopy imaging (5 slides per sample on average). Analysis of all samples was carried out by staff histologists of the “Municipal Clinical Hospital No. 1”, we used the diagnoses given to the patients. Some samples were independently analyzed at the Railroad Clinical Hospital (JSC Russian Railways, Novosibirsk) by an independent expert with work experience of more than 20 years.
RNA isolation
500 μL of guanidine lysis buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate, 0.3 % sarkosyl, 3 % DTT aliquoted in oxygen-free atmosphere, supplied by Vector-Best, Russia) was added to 50 mg of tissue. The sample was vigorously mixed and left in a thermal shaker for 15 min at 65 °C. Next, the tube was centrifuged at 10000 g for 2 min, the supernatant was transferred into new vials, followed by addition of an equal volume of isopropanol. Reaction was thoroughly mixed and left at room temperature for 5 min. After centrifugation for 10 min at 12000 g, the supernatant was discarded, and the pellet was washed with 500 μL 70 % ethanol and 300 μL acetone. Finally, the RNA was dissolved in 200 μL of deionized water. If not analyzed immediately, RNA preparations were stored at −20 °C until further use.
miRNA detection
To quantify miRNA, we followed the protocol published by Chen and co-authors in 2005 as it allows highly sensitive and specific identification of mature miRNAs. This protocol includes reverse transcription of mature miRNA using long stem-loop primer, which is followed by detection of cDNA via RT-qPCR [23]. Reverse transcription reactions were set individually for each miRNA to be quantified. The obtained cDNA was used for further PCR analysis immediately.
Synthetic analogs of miRNAs
Synthetic analogs of miRNAs were ordered from Biosan (Russia) and stored frozen in TE at −20 °C until needed. When used as controls, miRNA analogs were dissolved in deionized water and added directly into RT reaction mix, omitting the purification/isolation steps.
Oligonucleotide primers and probes
All oligonucleotides, including dual-labeled probes, were produced by Vector-Best (Russia). Oligonucleotide sequences were designed using online software tool PrimerQuest (Integrated DNA Technologies, USA). Several sets of primer and probe combinations were designed for each miRNA, and those showing high reverse transcription and PCR efficiencies were used for all downstream analyses. Efficiency of reverse transcription was assessed using quantification cycle (Cq) values obtained on synthetic miRNA analogs with known concentrations. Amplification efficiency (E) for each primers/probe combination was calculated by plotting a calibration curve over a series of RNA dilutions, with RNA isolated from clinical samples showing high levels of the miRNA of interest. E values ranged from 83.5 to 98.5 % for different miRNA amplifications. Sequences of primers and the probe are listed in Additional file 2. Sets of oligos for miR-146b, −181b −221 detection were validated by commercially available reagents TaqMan MicroRNA Assays (Applied Biosystems, USA). Within the linear range, the difference between Cq of our systems and Applied Biosystems not exceed 2. The difference in the efficiency of the reaction did not exceed 3.5 %.
Reverse transcription
Total volume of each reaction was 30 μL. Reaction mix contained 3 μL of RNA preparation, 21.6 % trehalose, 1x RT buffer (Vector-Best, Russia), 0.4 mM of each dNTP, 1 % BSA, 100U M-MLV reverse transcriptase (Vector-Best, Russia), 0.2 μM of appropriate RT primer. Reaction was incubated for 15 min at 16 °C and 15 min at 42 °C, which was followed by heat inactivation for 2 min at 95 °C. 3 μL of RT mix was used per one RT-qPCR reaction.
Real-time PCR
Real-time PCR was performed using CFX96 thermal cycler (Bio-Rad Laboratories, USA). All reactions were set up manually. Total volume of each reaction was 30 μL and encompassed 3 μL of cDNA, 1x PCR buffer (Vector-Best, Russia), 0.4 mM of each dNTP (Biosan, Russia), 1 % BSA, 1U Taq polymerase (Vector-Best, Russia) pre-mixed with active center-specific monoclonal antibody (Clontech, USA), 0.5 units of uracil-DNA glycosylase (Vector-Best), 0.5 μM of each primer and 0.25 μM of dual-labeled probe. PCR cycling conditions were as follows: 2 min incubation at 50 °C, pre-denaturation step at 94 °C – 2 min, followed by 50 cycles of denaturation (94 °C for 10 s), annealing and elongation (60 °С for 20 s).
U6 snRNA served as an internal normalization control because it is most commonly used for this purpose in studies on the expression of miRNA [24]. Fold change in expression of each miRNA in the tumor vs normal tissue was calculated using the following formula which takes amplification efficiencies of each PCR into account [25]:
$$ \frac{C_{tumor}}{C_{norm}}=\frac{\frac{{\left(1+{E}_{U6}\right)}^{Cq,U6(tumor)}}{{\left(1+{E}_{miR}\right)}^{Cq,miR(tumor)}}}{\frac{{\left(1+{E}_{U6}\right)}^{Cq,U6(norm)}}{{\left(1+{E}_{miR}\right)}^{Cq,miR(norm)}}} $$
E stands for amplification efficiency of miRNA or U6 internal control, and Cq - quantification cycle.
Reactions with Cq above 37 were considered as negatives. All experimental runs included NTCs (non-template controls) for each miRNA analyzed. NTC’s were run in triplicates. In our hands and with the equipment used, the Cq values for NTC’s always exceeded 37.
For each patient, miRNAs were profiled in 3 different samples from thyroid tumor tissue and in 3 different samples of matching adjacent non-tumor tissue; average values were taken into analysis.
Detection of BRAF(V600E)
Detection of BRAF(V600E) mutation was performed using allele-specific PCR with dual-labeled probe. Sequences of primers and the probe are listed in Additional file 2. PCR cycling conditions were as follows: pre-denaturation step 95 °С – 2 min, followed by 50 cycles of denaturation (94 °С, 10 s), annealing and elongation (60 °С, 15 s). Sensitivity of mutant allele detection, as assessed using samples with known wild-type and mutant sequences, was 0.75 % (data not shown). In cases where a mutation was detected, but no histology report stated “papillary carcinoma,” the presence of the mutation was confirmed by massive parallel sequencing on the platform 454 Junior (Roche).
Sequencing
Design of libraries of amplicons and sequences of primers for analysis with Roche GS Junior
The amplification of the BRAF gene was carried out in two rounds. In the first round fusion primers were used, consisting of sequences flanking BRAF V600E mutation (GATCCAGACAACTGTTCAAAC and ATCTCATTTTCCTATCAGAGCAA), and attached to their 5′-ends adaptor sequences “U13” (GCGGTCCAAAAGGGTCAGT) and “U9” (TTAATATTGCCACGGGCCTA). In the second PCR round fusion primers were used, consisting of auxiliary sequences of the Junior platform, and “U13” and “U9”, located at their 3′ends.
Sequencing
The sequencing was performed on the GS Junior instrument using Titanium kits and following the standard 200 nucleotide flows protocol according to the manufacturer’s recommendations.
Data analysis
The initial data analysis was carried out using software of the manufacturer (GS Run Processor, Roche), according to the preconfigured set of filters “Amplicons”, and then using application software Amplicon Variant Analyzer v. 3.0 (Roche).
Detection of RET-PTC1 translocation
Detection was performed using Real-time PCR combined with reverse transcription reaction in a single tube. Total volume of each reaction was 30 μL. Reaction mix contained 3 μL of RNA preparation, 16.7 % trehalose, 1x RT-PCR buffer (Vector-Best, Russia), 0.4 mM of each dNTP, 1 % BSA, 100U M-MLV reverse transcriptase (Vector-Best, Russia), 1U Taq polymerase (Vector-Best, Russia) pre-mixed with active center-specific monoclonal antibody (Clontech, USA), 0.5 μM of each primer and 0.25 μM of dual-labeled probe. RT-PCR protocol: incubation at 45 °С - 30 min., heating at 95 °С - 2 min., 50 cycles of denaturation at 94 °С - 10 s, annealing and elongation: 60 °С - 20 s. Sequences of primers and the probe are listed in Additional file 2.