Data Collection
The sample collection of this study was performed between March 2017 and July 2018 in Imam Reza hospital, Tabriz, Iran. Cytopathology reports of all patients who had undergone fine needle aspiration (FNA) from outpatient endocrinology clinic between March 2017 and July 2018 were analyzed, 40 samples with a definite diagnosis of PTC and 40 control samples from non-cancerous adjacent tissue of same patient were selected.
In this study, the control non-cancerous tissues is confirmed by H&M staining followed by microscopic evaluation. The experimental procedures described below, was performed in molecular laboratory setting in December 2020 at Nemooneh Medical Laboratory, Tabriz, Iran.
The exclusion criteria of our study were history of other thyroid diseases (due to the potential role of disrupted autophagy), previous chemotherapy or radiotherapy and diagnosis of other simultaneous malignancies.
Patient and Samples Information
This retrospective study excluded patients who had comorbid disorders, history of malignancy, contraindications for FNA and a family history of thyroid cancers.
Thyroid surgery samples including tumor and non-transformed adjacent tissues were obtained and underwent routine tissue collection procedure. After rinsing with normal saline, they were transferred to cryotubes and immediately preserved in a liquid nitrogen tank, then all samples were transferred to -70 degree freezer.
RNA Extraction
Trizol reagent was used to extract total RNA (Ambion life technologies, UK). The key components of this reagent are guanidiniumthiocyanate and phenol, which denature proteins and prevent RNase's unwanted effects on RNA material.
To extract total RNA, 300 mg of tissue was grinded using a porcelain mortar and pestle under liquid nirtrogen, homogenized tissue was transferred to 700 ul Trizol reagent directly in a microtube. The procedure followed by adding 200ul chloroform and centrifugation according to the manufacturer protocol (MAN0001271). The upper aqueous phase was transferred to a new microtube, total RNA purified by adding isopropyl alcohol, absolute ethanol, and 70% ethanol serially, separating by centrifugation steps. Purified RNA (ng/ul) quantity and possible protein or mineral residue contamination (extract quality) evaluated by Nanodrop device (Spectrophotometer 2000/2000c).
cDNA Synthesis
Two micrograms of purified mRNA was converted into cDNA by Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) in the 5Xbuffer, dNTP, and random hexamer primers, according to the manufacturer protocol (Lot N:cs0025). Steps installed for specific miRNAs and housekeeping miRNA by stem-loop technology, Biomir high sensitivity microRNA kit (Zistroyesh. Co).
Quantitative Real-Time PCR
To define the expression levels of genes, the quantitative Real-Time PCR (qPCR) technique was used. Gene-specific primer mix with PCR Master of Hot Start Taq DNA Polymerase and SYBR green was used for each gene (AnaCell).
The fluorescence emission variation of each gene in qPCR assay matched the quantity of mRNA or miRNA fragments in the sample Ct (cycling time) values in results that point on the qPCR curve which cuts off the threshold line.
qPCR amplifications program includes an initial 15 minute hold at 95° to trigger the Hot Star Taq Master Mix Taq polymerase followed by 20 seconds at 95° and an annealing-extension step of 60 seconds at 60° for 35–40 cycles with a final melt analysis step of 55°-95°.
Data Analysis
In this study, we tried to determine miR-182-5p, miR-183-5p, and miR-30d-5p as potential biomarkers in autophagy so our attempt to measure FOXO1 mRNA was considered as the extrinsic goal. To optimize data reproducibility, all tests were performed in triplicate format and normalized by efficiency index. Evaluating and analyzing Ct data has been done by the ΔΔCt method, first Ct data (Ct target gene-Ct ref housekeeping gene; treated sample)-(Ct target gene- Ct ref gene; untreated control) - the resulting ΔΔCT value be used as the exponent of power 2; (2^ -ΔΔCt) which is a conventional method for the expression value fold change variation [26]. Ct values were used to compare target and reference genes expression levels and fold changes were the reflection of a relative quantification.