Oligo dt 12 18 primer

Total RNA was extracted from the tumor metastases and SK-OV-3 cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. One microgram of total RNA was converted to cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo-(dT)_12–18 primers (Invitrogen) according to the manufacturer's instructions. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed in a 20 μl reaction mixture containing 1 μl cDNA, 10 μl SYBR Premix EX Taq (Takara Bio, Otsu, Japan), 0.4 μl Rox reference dye (50 ×, Takara Bio), and 200 nM primers for each gene. The primer sequences were: GABRP (forward), 5′-CTCGATTCAGTCCCTGCAAGA-3′ GABRP (reverse), 5′-GTGCGGGACCCGATCAT-3′ GAPDH (forward), 5′-AATCCCATCACCATCTTCCA-3′ and GAPDH (reverse), 5′-TGGACTCCACGACGTACTCA-3′. The reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) at 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s and a single cycle of 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s to generate dissociation curves. All PCR reactions were performed in triplicate, and the specificity of the reaction was determined by melting curve analysis. Comparative quantification of each target gene was performed based on cycle threshold (C_t) normalized to GAPDH using the ΔΔC_t method.

Because we inserted human fibroblasts and bFGF into the nude mice, the gene expression of collagen type I in the implanted materials was examined using RT-PCR with human-specific primers. The results were normalized to the mRNA level of human GAPDH.
Total RNA was extracted by adding 0.5 ml of TRIzol® reagent (Invitrogen, Life Technologies, USA) to N₂-frozen nude mice tissues. Each μg of RNA was subjected to cDNA synthesis by using SuperScript™ Reverse Transcriptase II (Invitrogen) and oligo (dT)12–18 primers (Invitrogen) in a 20 μl reaction volume according to the manufacturer’s instructions, with the additional step of removing the RNA complementary to the cDNA using E. coli RNase H (Invitrogen). One microliter of each cDNA was then subjected to polymerase chain reaction (PCR) according to the following amplification profile: predenaturation at 94 °C for 40 s, amplification (denaturation at 94 °C for 40 s; annealing at 60 °C for 40 s; extension at 72 °C for 1 min) for 30 cycles, and a final extension at 72 °C for 10 min in a DNA thermal cycler (model PTC-200, MJ Research, Inc., MA, USA). For each of the PCR products, 10 μl was electrophoresed on a 1.5% agarose gel in the presence of ethidium bromide and visualized by the Gel Documentation System (Vilber Lourmat, France).

Chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA, USA), unless otherwise stated. RNA polymerase, RNase-free DNase, and pGEM-T easy vector were purchased from Promega (Madison, WI, USA). Taq DNA Polymerase, Phusion High-Fidelity DNA Polymerase, and restriction endonucleases were purchased from New England BioLabs (Beverly, MA, USA). M-MLV reverse transcriptase, oligo(dT)_12–18 primers, and TRIzol^® Reagent were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA, USA). SMART™ RACE cDNA Amplification Kit was purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA). mMESSAGE mMACHINE kit was purchased from Applied Biosystems/Ambion (Austin, TX, USA). IGF peptides were purchased from GroPep (Adelaide, SA, Australia) and IGFBP-3 and BMP-2 were from R&D systems (Minneapolis, MN, USA). The anti-GFP antibody was purchased from Torrey Pines Biolabs Inc. (East Orange, NJ, USA). Anti phospho-Smad1/5/8 antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Total anti-Smad1/5/8 antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Digoxigenin and anti-Digoxigenin-POD antibodies were purchased from Roche (Indianapolis, IN, USA).

RNA extraction and real-time PCR were performed for the following genes: Angiopoietin-1 (Ang1), VEGF, VEGF receptor 2 (VEGFR-2), tunica interna endothelial cell kinase 2 (Tie-2) and ovine ribosomal protein S15 (ovRPS15). In brief, total RNA was extracted from snap-frozen RLL lung tissue by Trizol (15596018; Invitrogen, Life Technologies, Carlsbad, CA)/chloroform extraction. Isolated RNA was DNAse treated to remove possible contamination with genomic DNA by use of the RQ1 RNase-Free DNase kit (M6101; Promega, Madison, WI) and afterwards reverse transcribed into cDNA using oligo(dT)_12-18 primers (18418–012; Invitrogen) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (28025–021; Invitrogen). Real-time PCR reactions were performed in duplicate within a LightCycler 480 Instrument (Roche Applied Science, Basel, Switzerland) using the SensiMix™ SYBR® No-ROX Kit (QT650; Bioline, London, UK) for 45 cycles. Primer sequences (Table 1) were designed or used from literature [26 (link), 27 (link)]. Results were normalized to the house keeping gene ovRPS15 and relative changes, over control values, were calculated.

Total RNA was isolated from transfected HTR-8/SVneo cells (L, S and sigalectin-3 group) using TRIzol (Applied Biosystems, Carlsbad, CA, USA), as suggested by the manufacturer. First-strand cDNA was synthesized from 1 µg of total RNA, using 0.5 μg of Oligo(dT) 12–18 primers (Invitrogen, Carlsbad, CA, USA), 250 µM of each dNTP and 200U of RevertAid reverse transcriptase (Fermentas, Vilnius, Lithuania). Real-time PCR was performed using the 7500 Real-Time PCR System (Applied Biosystems, Carlsberg, USA). The reaction mixture contained 1 µl of cDNA, 5 µl 2x SYBR® Green PCR Master Mix (Applied Biosystems, Carlsberg, USA) and specific forward and reverse primers in a final concentration of 0.5 µM. Reactions were run at 95 °C for 10 min, followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. Melting curve analysis was performed to verify amplification specificity. Expression levels of LGALS3, LGALS1, LGALS8, ITGA1, ITGA5, ITGB1, MMP2 and MMP9, were normalized to the housekeeping gene GAPDH. Calculations were made by the comparative ΔΔCt method^{72 (link)}. The sequences of specific primers are given in supplemental material (STable 1).