Gene expression levels of IL-1β and NLRP3 were determined by real-time PCR. BMDCs from WT and TLR2-deficient mice were infected with P. nigrescens at an MOI of 100 for 2 h and RNA was extracted using the easy-BLUETM Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). cDNA was synthesized from 0.1 μg of RNA using ReverTra Ace® qPCR RT Master Mix (TOYOBO Bio-Technology, Osaka, Japan) according to the manufacturer's instructions. PCR was performed using the QGreenTM 2X SybrGreen qPCR kit (Qiagen GmbH, Hilden, Germany). GAPDH was used for normalization of expression levels. The following primer sequences were used: IL-1β (IL-1β forward 5'-GATCCACACTCTCCAGCTGCA-3', IL-1β reverse 5'-CAACCAACAAGTGATATTCTCCATG-3'); NLRP3 (NLRP3 forward 5'-ATGGTATGCCAGGAGGACAG-3', NLRP3 reverse 5'-ATGCTCCTTGACCAGTTGGA-3'); AIM2 (AIM2 forward 5'-AACCCAAGCAAAACAAAGTG-3', AIM2 reverse 5'-GCTACAAGGTCCAGATTTCAAC-3'); ASC (ASC forward 5'-TCACAGAAGTGGACGGAGTG-3', ASC reverse 5'-TCATCTTGTCTTGGCTGGTG-3'); Caspase-1 (Caspase-1 forward 5'-GCCCACTGCTGATAGGGTGA-3', Caspase-1 reverse 5'-CCCGGGAAGAGGTAGAAACG-3'); NLRC4 (NLRC4 forward 5'-ACGCAGGCAAAACACTCATA-3', NLRC4 reverse 5'-TCGTTTCTCAAGCCAATTCC-3'); and GAPDH (GAPDH forward 5'-CGACTTCAACAGCAACTCCCACTCTTCC-3', GAPDH reverse 5'-TGGGTGGTCCAGGGTTTCTTACTCCTT-3'). PCR amplification was performed using a three-step protocol of 95°C for 10 seconds followed by 40 cycles of 58°C for 15 seconds, and 72°C for 20 seconds in a Rotor-Gene Q real-time PCR system (Qiagen).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.