Micro amp optical adhesive films

Total RNA was extracted using deparaffinization solution (Qiagen, cat. QG19093, Hilden, Germany) and miRNeasy FFPE Kit (Qiagen, cat QG217504) according to manufacturer’s instruction from 5 sections (10 µm each) of FFPE (formalin fixed paraffin embedded) samples of murine and human colons. Then, RNA (0.8 μg) was reverse transcribed via Reliance Select cDNA Synthesis KT (Bio-rad, cat. 12012801, Hercules, CA, USA). cDNA was subjected to real-time PCR, using the iQ™ Multiplex Powermix, (Bio-rad, cat. 1725849), together with gene-specific PrimePCR™ Probe Assays (Bio-rad, Table 1) for detecting expression of human genes or using PowerUp SYBR Master Mix (TermoFisher) with gene-specific primers (Themo fisher, Table 2) for detecting expression of mouse genes. Reactions were set up in Primo^® FrameStar^® 96-well PCR plates (Euroclone, cat. ECPCR0770C, Pero, Italy), which were sealed with MicroAmp™ optical adhesive films (Applied Biosystems, cat. 4360954, Waltham, MA, USA). The thermal profile of Cielo 6 qPCR system (Azure Biosystem, Dublin, CA, USA) was set as follows: 2 min at 95 °C, then 45 cycles of 15 s at 95 °C and 20 s at 60 °C, along with a final post-read stage of 30 s at 60 °C.

Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin-streptomycin cocktail, amphotericin B, sodium bicarbonate, TRIzol reagent, Hoechst 33342, and Escort transfection reagent were purchased from Sigma-Aldrich (USA). Fetal bovine serum was purchased from Gibco (USA). Restriction enzymes, Turbofect, M-MuLV Reverse Transcriptase kit, Hi-fidelity PCR enzyme mix, FastAP thermo sensitive Alkaline Phosphatase and the INSTAclone TA-cloning kit were purchased either from MBI Fermentas (USA) or from NEB (USA). QIAquick gel extraction kit was purchased from Qiagen (USA). Protein A-CL agarose bead resin was purchased from Bangalore Genei (India). 2X SYBR Green PCR master mix, micro-amp Fast 96-well reaction plates (0.1 ml) and micro-amp optical adhesive films were purchased either from Applied Biosystems (USA) or Kapa Biosystems (USA). For western blotting, Immobilon-P PVDF membrane was purchased from Merck-Millipore (USA). X-ray films, developer, and fixer were from Kodak (USA).

Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin-streptomycin cocktail, amphotericin B, sodium bicarbonate, fetal bovine serum, TRIzol reagent, primers, Hoechst 33342, doxorubicin, thymidine and Escort^TM transfection reagent were purchased from Sigma-Aldrich (USA). Zeocin was obtained from Invitrogen (USA). Restriction enzymes, Turbofect, M-MuLV Reverse Transcriptase kit, Hi-fidelity PCR enzyme mix, FastAP thermo sensitive Alkaline Phosphatase and the INSTAclone TA-cloning kit were purchased either from MBI Fermentas (USA) or from NEB (USA). QIAquick gel extraction kit was purchased from Qiagen (USA). Protein A-CL agarose bead resin was purchased from Merck (India) and Protein-G fast flow bead resin was purchased from Merck-Millipore (USA). 2X SYBR Green PCR master mix, micro-amp Fast 96-well reaction plates (0.1 ml) and micro-amp optical adhesive films were purchased from Applied Biosystems (USA). 2X fast SYBR Green PCR master mix was purchased from Kapa Biosystems (USA). Dual luciferase assay kit was purchased from Promega (USA). For western blotting, Immobilon-P PVDF membrane was purchased from Merck-Millipore (USA). X-ray films, developer, and fixer were from Kodak (USA).