Trizol ls kit

Total RNA was extracted from tumor tissue and plasma samples by Trizol LS kit (TaKaRa, Japan), and RNA was subjected to reverse transcription by utilizing the PrimerScript Real-time reagent kit (TaKaRa, Japan). Subsequently, miR-146 a/b expressions were quantitated by SYBR Premix Ex TaqTM II (TaKaRa, Japan). The primer sequences (BGI, China) for the primer source were performed as follows: hsa-miR-146a-5p RT primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACCCATG-3′; hsa-miR-146a-5p Forward primer: 5′-ACACTCCAGCTGGGTGAGAACTGAATTCCATG-3′; hsa-miR-146a-5p Revert: 5′-TGTCGTGGAGTCGGCAATTC-3′; hsa-miR-146b-5p RT primer; 5′-ACACTCCAGCTGGGTGAGAACTGAATTCCATG-3′; hsa-miR-146b-5p Forward primer: 5′-ACACTCCAGCTGGGTGAGAACTGAATTCCATA-3′; hsa-miR-146b-5p Revert: 5′-TGTCGTGGAGTCGGCAATTC-3′. The expression levels of miR-146a/b were calculated by using the 2^-△△t method and U6 served as the internal reference.

A panel consisting of 13 pro‐angiogenic miRNAs was selected in this study based on a previous report 16. Total RNA was extracted from plasma samples with a TRIzol LS kit (Takara, Japan). RNA was reverse transcribed using the One Step Primer Script miRNA cDNA Synthesis Kit (Takara, Japan), and the expression levels of these 13 candidate miRNAs were quantitatively analyzed using SYBR Premix Ex TaqTM II (Takara). U6 expression, which served as an internal reference, was used to normalize the expression of miRNAs, and the expression levels of these candidate miRNAs were then calculated with the 2^−∆∆t method.

Fourteen candidate pro‐angiogenic miRNAs were selected by analyzing a previous study.17 Total RNA was extracted from the plasma sample by Trizol LS kit (Takara, Japan). The reverse transcription of RNA was conducted by One Step Primer Scrip miRNA cDNA Synthesis Kit (Takara). qPCR procedures were conducted as follows: all reactions were incubated on a 96‐well plates at 95°C for 5 minutes, followed by 40 cycles of amplification, and each cycle included denaturation at 95°C for 5 seconds, annealing at 61°C for 15 seconds, and extension at 72°C for 30 seconds. U6 expression was used as the internal reference to normalize miRNA expressions, and the expressions of these candidate miRNAs were calculated by 2^−ΔΔct method. The primers used in this study were listed in Table 1.