2.4. Analysis of miRNA Gene Expression with Reverse Transcription and RT-qPCR

qRT-PCR was performed on total RNA using the four miRNAs and internal control (IC) primers for standardization. The reaction solution (total RNA, primers, 2X qRT-PCR master mixture, and ddH₂O) was added to each of the four prepared tubes. qRT-PCR was performed under the following conditions: 50°C for 15 minutes (1 cycle) ⟶ 95°C for 10 minutes (1 cycle) ⟶ 95°C for 10 seconds, and 65°C for 20 seconds (40 cycles). A Bio-Rad CFX96 Dx system (Bio-Rad, Hercules, CA, USA) was used for genetic analysis. The primer sequences used for PCR were as follows (X in the primer sequence represents inosine): miR-1246 ({"type":"entrez-nucleotide","attrs":{"text":"NR_031648.1","term_id":"269847260","term_text":"NR_031648.1"}}NR_031648.1) primer sequences, forward 5′-TCT CTXXXT GAA GTA GGA CTG GGC AGA GA-3′ and reverse 5′-CTC AAXXXT GTT TGC AAT AGC CCT TTG AG-3′; miR-202 ({"type":"entrez-nucleotide","attrs":{"text":"NR_030170.1","term_id":"262205198","term_text":"NR_030170.1"}}NR_030170.1) primer sequences, forward 5′-GGC CAXXXG CAT ATA CTT CTT TGA GGA TCT GGC C-3′ and reverse 5′-CAT GGXXXG ACC GCC CCG TTT TCC CAT G-3′; miR-21 ({"type":"entrez-nucleotide","attrs":{"text":"NR_029493.1","term_id":"262205659","term_text":"NR_029493.1"}}NR_029493.1) primer sequences, forward 5′-CAGTCXXXG TCG GGT AGC TTA TCA GAC TG-3′ and reverse 5′-CAG TCXXXC AGA CAG CCC ATC GAC TG-3′; miR-219B ({"type":"entrez-nucleotide","attrs":{"text":"NR_039815.1","term_id":"337756649","term_text":"NR_039815.1"}}NR_039815.1) primer sequences, forward 5′-ACA TCXXXG GAG CTC AGC CAC AGA TGT-3′ and reverse 5′-GTT TGXXXG CGC CAC TGA TTG TCC AAA C-3′; and human hemoglobin subunit beta gene (MK_476504.1) primer sequence, forward 5′-GGA CAX XXC ACT AAG CTC GCT TTC TTG CTG TCC-3′ and reverse 5′-GGA TAX XXG ATG CTC AAG GCC CTT CAT AAT ATC C-3′. Human hemoglobin subunit beta was used as the reference gene for qRT-PCR. The basis for selecting the reference gene was the correlation between RNA concentrations and gene expression (Ct value), which is the result of qRT-PCR analysis. The reference gene was most accurate in the Ct value range of 25 to 30 and was not different between the healthy control and breast cancer patient groups. The RNA concentrations used in the experiment were standardized to those of the reference gene. Cycle threshold (Ct) was defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeding the background level). Delta (dCt) was defined as Ct (reference gene)–Ct (target gene).