Briefly, 2 μg of total RNA that had been previously DNased following manufacturer instructions (Turbo DNase, Ambion) was mixed with 0.5 μg of random hexamer and RNase-free water (to bring up the volume to 12 μl), heated at 70°C for 10 min, and cooled on ice.
cDNA synthesis was conducted as followed with the SYBR® ExScript™ RT PCR kit (Takara Biotechnology Co. Ltd., Dalian, China) according to manufacturer's instruction: 500 ng total RNA was mixed with 2 μl of 5 × ExScript™ RTase buffer, 0.5 μl of dNTP mixture, 0.5 μl of random hexamers, 0.25 μl of ExScript™ Rtase, and 0.25 μl of RNase inhibitor in a total volume of 10 μl.
The reaction was performed according to the manufacturer's instructions, briefly, 30 μl of RNA was mixed with 3 μl of Buffer A, 0.5 μl RNAsin® Ribonuclease inhibitor (20 40 U/μl, Promega) and 1 μl Terminator (1 U/μl).
The prepackaged grout was mixed per the manufacturer's instructions with a fluidity of 180 270 mm for easy operation.
Each serum (100 μL) was mixed with sample diluent according to the manufacturer's instructions.
The urine samples studied were processed according to instructions for non mucoid fluids: samples were mixed with a Cytolyt® solution containing methanol, mucolytic and hemolytic agents and were then centrifuged at 600 G for 10 minutes.
Tubes were mixed by inverting 5-8 times, following the manufacturer's instructions, immediately after sample collection.
Follow the instructions that come with your box kit to determine how the color should be mixed.
Powdered flux must be mixed into a paste or liquid form first. Check the instructions for details.
After 6-h incubation, cells were mixed with test reagents and luminescence was read according to the manufacturer's instructions.
