Microamp optical adhesive film

Protein thermal shift was detected according to the previous description and the manufacturer’s method using HSP90 recombinant protein (1 μg/μL/reaction) and different concentrations of Apl-1 as ligands [21 (link)]. The thermal shift buffer (5 μL) was added, 8X Sypro Orange fluorescent dye (2.5 μL) was added, and the final volume of 20 μL was attained with distilled H₂O. Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, MA, USA) was used to spot the tested compounds at different concentrations on MicroAmp Optical Adhesive Film (Applied Biosystems, Foster City, CA, USA). The reactants were sealed and mixed. A Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect different concentrations of Apl-1 samples on MicroAmp Optical Adhesive Film (Applied Biosystems, Foster City, CA, USA), and the reactants were sealed and mixed. The plate temperature was heated from 25 to 99 °C at a heating rate of 1 °C/min. The detection was undertaken in the Step One Plus Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA). Displacement software (version 1.3, Applied Biosystems, Foster City, CA, USA) was used to calculate the melting temperatures (Tm) and to establish the thermal curve. The determination of Apl-1 was carried out at different concentrations in triplicate.

For real-time quantification of target gene expression, one-step real-time Polymerase Chain Reaction (RT-PCR) was performed using FastStart Universal SYBR Green Master (ROX) in a StepOnePlus^™ Real-Time PCR System (Life Technologies-Invitrogen, California, U.S.A.). A fragment of the NOMO1 gene was amplified from the DNA of patients and controls using the following primers: F: 5′-agctccatgtggatggagtc-3′ and R: 5`-acggatgaagtacagagttc-3. As internal control, the 36b4 gene was amplified from the same DNA using the primers: F: 5′-cagcaagtgggaaggtgtaatcc-3′ and R: 5′-cccattctatcatcaacgggtacaa-3.
Ten μl RT-PCR of a mix containing 15 ng of total DNA, 1 μl of the primer dilution, 4 μl FastStart Universal SYBR Green Master and 4 μl H₂O were used for amplification. One-step RT-PCR reactions were carried out in 96-well optical reaction plates, covered with MicroAmp^® Optical Adhesive Film (Life Technologies-Invitrogen, California, U.S.A). Cycling was as follows: 10 minutes at 95°C followed by 40 cycles of 95°C for 15 seconds, 58°C for 45 seconds and 72°C for 15 seconds. RQ Manager software was used to analyse the values.
The comparative Ct method (2^−ΔΔCt) was used to calculate the relative expression levels of each amplicon. RT-PCR specificity of each PCR reaction was verified by melting curve analysis.