Abi 7900 thermocycler

Real-time quantitative polymerase chain reaction (PCR) analysis was performed using SYBR Premix Ex Taq (TaKaRa) for mRNAs and SYBR Premix Ex Taq II for miRNAs in an ABI 7900 thermocycler (Applied Biosystems, CA, USA). Gapdh served as a reference gene for analyzing gene expression levels. The real-time PCR data were analyzed using the 2^−ΔΔCT method. The primers were synthesized by Sangon (Shanghai, China). The primer sequences for real-time PCR are shown in Supplementary Tables S1 and S2.

Equal amount of freshly purified HeLa nucleolus suspension was either heated to 95 °C (“heat-denatured nucleolus”) for 5 min or incubated on ice for 5 min (intact nucleolus). The nucleoli were then subjected directly to reverse transcription using SuperScript® III Reverse Transcriptase (Invitrogen) and random hexamers according to the manufacturer’s instructions. QPCR was performed using SYBR Green (Atila Biosystem) and specific primer pairs. Amplification was conducted for 38 cycles at 94 °C for 10 s and at 60 °C for 1 min each using an ABI7900 thermocycler (Applied Biosystems). ΔCt represents Ct heat-denaturated nucleoli – Ct non-denatured nucleoli. The primer sequences used were as follows: RNU44: 5′-CCTGGATGATGATAGCAAATGC-3′ and 5′-GAGCTAATTAAGACCTTCATGTT-3′; RNU48: 5′-AGTGATGATGACCCCAGGTAA-3′ and 5′-GTGATGGCATCAGCGACACA-3′; HBII-239: 5′-GAAGCAGTGGGAGTGGAGAA-3′ and 5′-TCAGCAGTTTGAGTGTCAGCA-3′; hY1: 5′-GGCTGGTCCGAAGGTAGTGA-3′ and 5′-GCAGTAGTGAGAAGGGGGGA-3′; hY3: 5′-GGCTGGTCCGAGTGCAGTG-3′ and 5′-GAAGCAGTGGGAGTGGAGAA-3′; U1: 5′-ATACTTACCTGGCAGGGGAGA-3′ and 5′-CAGGGGGAAAGCGCGAACGCA-3′; U2: 5′-ATCGCTTCTCGGCCTTTTTG-3′ and 5′-TCCTATTCCATCTCCCTGCTC-3′. Primers for miR-206, miR-23a and miR-100 were from Applied Biosystems (TaqMan miRNA Assays ID 000510, ID 000399 and ID 000437).

The mRNA expression levels in tissues and cells were determined by RT-qPCR. The factors involve MON1B in colon cancer tissues and different cell lines, as well as cell invasion-related factors after MON1B was silenced in LoVo cells. Total RNA was extracted with TRIzol Reagent (Invitrogen, USA), and 1 μg RNA was reversely transcribed to cDNA using EN-QuantiTect Reverse Transcription (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocols. PCR amplification was conducted using Fast SYBR Green Master Mix (Applied Biosystems, USA) and detected using an ABI 7900 Thermocycler (Applied Biosystems, USA): 25 s at 95°C for initial denaturation, 40 cycles setting (20 s at 95°C, 20 s at 57°C, 30 s at 72°C), and 10 min at 72°C for final extension. The primer sequences of MON1B, matrix metalloproteinases (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2, metastasis-associated antigen (MTA-1), NF-κB p65, IκB, Chemokine receptor type-4 (CXCR-4), and β-actin are displayed in Table 2.