Dtlite real time pcr system

Expression of PR and NF2 mRNA was assessed from serum using real time quantitative polymerase chain reaction (RT-qPCR) following standardized protocol. All procedures were performed by trained technician under direct supervision of experienced expert (D.S.H). In principal, a total of 5 ml of venous blood were taken from brachial vein and collected in the tubes containing EDTA. Total RNA was extracted from whole blood using the Geneaid Blood/Cell Total RNA Mini Kit (Geneaid Biotech, Taiwan). The total RNA was subjected to reverse transcription followed by qPCR analysis in 48-well with the use of KAPA SYBR® FAST One-Step Universal Kit (Sigma-Aldrich, St. Louis, MO, USA) and a DTlite Real-Time PCR System (DNA–Technology, Russia). The amplification results were analyzed with the use of DTlite Real-Time PCR System Software (DNA–Technology) and were normalized by the corresponding amount of GAPDH mRNA. Primer sequences for qPCR (forward and reverse, respectively) were as follows: GAPDH, 5′-GCATCCTGGGCTACACTGAG-3′ and 5′TCCACCACCCTGTTGCTGTA-3′; PR, 5′-AGCTCATCAAGGCAATTGGTTT-3′ and 5′-ACAAGATCATGCAAGTTATCAAGAAGTT-3; and NF2, 5′-CCCCCAACTCCCCTTTCC-3′ and 5′-AGCCCTTTAGCCCCCCTG-3′.

All the quantitative real-time PCR assays containing the primer and probe mix were purchased from Gene-All Hybrid and utilised according to the manufacturer's instructions. Real-time PCRs were done using One-Step qRT-PCT with KAPA SYBR FAST Universal. Reactions of the PCRs were carried out using the forward primer GAPDH (5′-GCA TCC TGG GCT ACA CTG AG-3′), CXCL12 (5′-GAT TGT AGC CCG GCT GAA GA-3), and CXCR4 (5′-AGC ATG ACG GAC AAG TAC C-3′) and the reverse primer GAPDH (5′TCC ACC CTG TTG CTG TA-3′), CXCL12 (5′ TTC GGG TCA ATG CAC ACT TGT-3), and CXCR4 (5′GAT GAT ATG GAC ACC CTT ACA C-3′). An individual reaction was performed using the DT-Lite Real-Time PCR System (DNA Technology) with reverse transcription at 42°C for 5 minutes, followed by enzymatic activation at 95°C for 3 minutes, denaturation for 1–3 seconds at 95°C, and elongation for up to 20 seconds at 60°C.
The normal tissue became the 1x sample, and all the other quantities were expressed as the n-fold difference relative to this tissue as the control. The expression rates of CXCL12 and CXCR4 were counted using the formula: 2^(−ΔΔCT), with ΔCT = PCR score of CXCL12 or CXCR4-PCR score GAPDH. ΔΔCT = ΔCT of tumour sample − ΔCT normal colon tissue [29 (link)].

Each PCR was performed in a 25 μL mixture composed of 5 μL of qPCRmix-HS SYBR (Evrogen, Moscow, Russia), 1 μL (0.2 μM) of the primer solution, 17 μL water (RNase-free), 1 μL cDNA. A Dtlite Real-Time PCR System (DNA-technology, Moscow, Russia) was used for amplification. The amplification process consisted of the initial 5 min denaturation at 95 °C, 40 cycles of 30 s denaturation at 95 °C, 20 s annealing at 60–62 °C, and 20 s extension step at 72 °C. The final extension was performed for 10 min at 72 °C. All the sequences were designed with FAST PCR 5.4 and NCBI Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi, accessed on 6 July 2023). The data were analyzed with Dtlite software (https://dna-technology.com/sites/default/files/dtprime_dtlite_v06_part_2.pdf, accessed on 6 July 2023) (DNA-technology, Moscow, Russia). The expression of the studied genes was normalized to gene encoding Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [92 (link),93 (link)]. Data were analyzed using Livak’s method [94 (link)].