This study had been approved by the Institutional Ethics Committee, All India Institute of Medical Sciences, Jodhpur. This study involved 35 T2DM patients aged 30-60 years, recruited from the Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, Jodhpur, Rajasthan. The American Diabetes Association (ADA) guidelines had been used to diagnose T2DM . Fasting blood glucose (FBG) ≥126 mg/dl, 2h OGTT ≥ 200 mg/dl and HbA1c ≥ 6.5% were diabetic. Also, 35 Non-T2DM controls were taken from healthy individuals aged 30-60 years. The Control group was confirmed to not have suffered from any Diabetes diseases as per the American Association of Diabetes. Those of age <30 years or with serious comorbid conditions including diseases of the lung, kidney, heart, and liver, or those with hematologic or immune disorders, pregnancy, type-1 diabetes, or malignancy were excluded from the study. The blood samples of T2DM patients and Non-T2DM controls were collected in the hospital after due informed consent.
From each participant, peripheral whole blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes (Greiner Bio-one, Germany). Total RNA was isolated within 2 hrs of sample collection. Serum samples were collected and basic investigations like HbA1c (glycated haemoglobin), lipid profile [TC (total cholesterol), TG (triglycerides), HDL-c (high density lipoprotein cholesterol), LDL-c (low density lipoprotein cholesterol)], hs-CRP (high sensitivity C-reactive protein), FBG (fasting blood glucose) was done.
RNA Extraction and Reverse Transcription
Total RNA, including miRNA, was isolated from 200 μl of whole blood samples using the Trizol LS reagent (Thermo Fischer Scientific, US)  following the manufacturer’s instruction. The quality and quantity of RNAs were assessed using a NanoDrop Onec (Thermo Fischer Scientific, US). All RNA samples were stored at -80oC until further processing. Reverse transcription (RT) was carried out using the TaqMan® Advanced miRNA cDNA Synthesis Kit (Thermo Fischer Scientific, US). cDNA synthesis was the multistep process which includes the poly(A) tailing reaction, in which 2μl of total RNA was mixed with 10X Poly(A) buffer (0.5μl), ATP(0.5μl), poly(A) enzyme(0.3μl), RNase-free water(1.7μl) to a final volume of 5μl. The reaction mixtures were then incubated at 37oC for 45 min, at 65oC for 10 min, and then held at 4oC. The second step was adaptor ligation reaction in which 5μl of poly(A) tailing reaction product mixed with 5X DNA Ligase buffer (3μL), 50% PEG 8000 (4.5μL), 25X Ligation Adaptor (0.6μL), RNA Ligase (1.5μL), RNase-free water (0.4μL) to a total volume of 15μL. Then this mixture was incubated at 16°C for 60 minutes and held at 4°C. Next step was reverse transcription (RT) reaction in which 15μL of adaptor ligation reaction product was mixed with 5X RT buffer (6μL), dNTP Mix (25mM each) (1.2μL), 20X Universal RT Primer (1.5μL), 10X RT enzyme Mix (3μL), RNase-free water (3.3μL) to a total volume of 30μl. The reaction mixture was incubated at 42°C for 15 minutes, stop the reaction at 85°C for 5 minutes, and held at 4°C. The final step for cDNA synthesis was a miR-Amp reaction in which 5μL of the RT reaction product mixed with 2X miR-Amp Master Mix (25μL), 20X miR-Amp Primer Mix (2.5μL), RNase-free water (17.5μL) to a final volume of 50μl. The reaction mixture was incubated at 95°C for 5 minutes for 1 cycle, denature at 95°C for 3 seconds for 14 cycles, anneal/extend at 60°C for 30 seconds, stop the reaction at 99°C for 10 minutes and held at 4°C. undiluted miR-Amp reaction product stored at –20°C until analysis. 1:10 dilution of cDNA template was done before using it in RT PCR (5μL of the miR-Amp reaction product to 45μL 0.1X TE buffer)
Micro-RNAs profiling by Real-Time PCR
The relative expression of miR-24-3p and miR-198 were assessed using the TaqMan Advanced miRNA Assays kit (Applied Biosystems; Thermo Fisher Scientific, US). Expression levels of miRNAs were determined using TaqMan MGB probes and TaqMan Universal PCR Master Mix II (2x) in triplicate. Diluted cDNA (5μl) was used as a template in a 20μl reaction mix containing 10μl of TaqMan® Fast Advanced Master Mix (2X), 1μl of TaqMan® Advanced miRNA Assay (20X) and 4μl RNase-free water (Applied Biosystems; Thermo Fisher Scientific, US). RT-qPCR reactions were run with Biorad CFX 96 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, US) 95°C for 20 seconds followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds. Triplicate measurements were obtained for each sample and mean were taken. The data were analyzed with the automatic threshold cycle (Ct) setting for assigning baseline and threshold for Ct determination. The relative expression of each miRNA was calculated using the 2-∆∆Ct method. The expression levels of miRNAs: miR-24-3p and miR-198 were normalized to miR-16-5p, as an internal control.
Data were presented as the mean ± standard deviation (SD) and median (IQR). Comparisons of anthropometric and biochemical data between cases and controls were analyzed by Mann Whitney U test. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic accuracy of miRNAs and the area under the curve (AUC) was reported for each miRNA. Probability (p) value <0.05 was considered significant. All statistics were performed using GraphPad Prism 8 software.