Trizol technique

Two-hundred and fifty-six BM samples were obtained from patients with MM during routine diagnostic procedures at the Peking University People’s Hospital. Mononuclear cells were isolated from BM samples using standard Ficoll-Hypaque density gradient centrifugation. RNA was extracted from mononuclear cells using the TRIzol technique (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized as previously described
[20 (link)].

RNA was extracted from hippocampal tissue via the Trizol technique (Invitrogen Life Technologies), which was then followed by cDNA reverse transcription facilitated by a kit from Takara (Tokyo, Japan). The subsequent quantitative real-time polymerase chain reaction (qRT-PCR) made it possible to ascertain the ΔCt value during the period of exponential amplification. The PCR reaction process was as follows: an initial pre-denaturation phase at 94 °C for 15 min, denaturation at 94 °C for a span of 10 s, and annealing/extension at 66 °C for 30 s, repeating this sequence for 40 cycles. Each sample underwent testing in triplicate using the ΔΔCt method for assessing shifts in gene expression, with adjustments made relative to GAPDH.
The primers implemented in the study included: GAPDH (forward 5′-ACTCCACTCACGGCAAATTC-3′, reverse 5′-TCTCCATGGTGGTGAAGACA-3′), IGF-1 (forward 5′-TGCCCTCAACCCCACTACTG-3′, reverse 5′-CGGTTGTCACTGGTTCATGTG-3′), CD86 (forward 5′-CATGGGCTTGGCAATCCTTA-3′, reverse 5′-AAATGGGCACGGCAGATATG-3′), iNOS (forward 5′-ACATCGACCCGTCCACAGTAT-3′, reverse 5′-CAGAGGGGTAGGCTTGTCTC-3′), CD206 (forward 5′-CTCTGTTCAGCTATTGGACGC-3′, reverse 5′-TGGCACTCCCAAACATAATTTGA-3′), and Arg-1 (forward 5′-CTCCAAGCCAAAGTCCTTAGAG-3′, reverse 5′-GGAGCTGTCATTAGGGACATCA-3′).

Using the TRIzol technique (Life Technologies Corp., Grand Island, NY, USA), total RNA was isolated from tumor tissue in accordance with the manufacturer’s protocol and stored at −80 °C for further analysis. To evaluate RNA concentration and purity, the Nanodrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used. Using cDNA archive kit (Applied Biosystems, Foster City, CA, USA), RNA was transformed into its complementary DNA. cDNA was utilized for quantitative PCR through SYBR Green (SYBR^® Premix Ex Taq™ II, TaKaRa, Dalian, China). Using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), the RT-PCR protocol included an initial denaturation step at 94 °C for 3 min, then 40 cycles for 45 s at 94 °C, then at their respective annealing temperatures for 30 s, and at 72 °C for 30 s followed by a 10-min extension step at 72 °C [71 (link)]. The expression of β-Actin served as the internal control, and the relative expression was determined [72 (link)]. The primer sequences of tested P53, NF-κB, miRNA-191-5p, and miR-543 as well as β-Actin are displayed in Table 6.