Ambion mirvana mirna isolation kit

Isolation of total RNA from cell lysates was performed using the Ambion^® miRVana miRNA isolation kit (Life Technologies). Quantitation of total RNA quantity and quality was determined using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Inc.). Complementary DNA (cDNA) was generated from 10 ng total RNA samples using the TaqMan^® miRNA reverse transcription kit (Life Technologies). qPCR was performed using 1.33 µl of cDNA and TaqMan^® microRNA assays (Life Technologies). Reactions were run in duplicate.

The miRNAs were isolated from 400-µL saliva samples using an Ambion mirVana miRNA isolation kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instruction. Complementary DNA (cDNA) was synthesized with a miScript II RT kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Finally, PCR array experiments were performed using a miScript miRNA PCR Array for Human Inflammatory Response and Autoimmunity and miScript SYBR Green PCR kit (Qiagen). The miScript miRNA PCR Array for Human Inflammatory Response and Autoimmunity identifies 84 miRNAs related to inflammatory response and autoimmunity. A total volume of 1,100 µL of PCR reaction mix was prepared (550 µL of 2× QuantiTect SYBR Green PCR master mix, 110 µL of 10× miScript universal primer, 340 µL of RNase-free water and 100 µL of template cDNA) before addition of 10 µL of reaction mix to each well of the miScript miRNA PCR Array. Optical adhesive film was then sealed onto the array. The array was centrifuged for 1 minute at 1,000 ×g at room temperature to remove bubbles. The PCR real-time cycler was programmed as follows: the initial activation step was carried out for 15 minutes at 95°C, followed by 40 cycles of denaturation (15 seconds, 94°C), annealing (30 seconds, 55°C), and extension (30 seconds, 70°C).