Tissues were frozen in cold QIAzol lysis reagent and homogenized using a handheld homogenizer. After centrifugation at 12,000g for 10 minutes, the supernatants were used for total RNA extraction using RNeasy Plus Universal Mini kit. For in vitro studies, cells were lysed in QIAzol lysis reagent and RNA was also extracted using RNeasy Plus Universal Mini kit. RNA samples were reverse transcribed into cDNA using SuperScript III Reverse Transcriptase or MultiScribe Reverse Transcriptase (Moloney murine leukemia virus reverse transcriptase, Invitrogen). qPCR was performed according to the manufacturer’s protocol (Applied Biosystems) by incubating cDNA samples with specified probes and primers of interest and TaqMan Universal PCR Master Mix II and measuring PCR amplification using the 7900HT Real-Time PCR System. Probes and primers for Hmox1 (Mm00516004_m1), Rela (NF-κB subunit p65; Mm00501346_m1), Socs1 (Mm01342740_g1), Socs3 (Mm00545913_s1), Stat3 (Mm01219775_m1), Stat1 (Mm01257286_m1), Nos2 (Mm00440502_m1), Irf1 (Mm01288580_m1), Irf3 (Mm00516784_m1), Irf8 (Mm00492567_m1), Slc40a1 (Mm01254822_m1), C3 (Mm01232779_m1), Cfb (Mm00433909_m1), Gapdh (Mm99999915_g1), and 18S (Hs99999901_s1) were commercially available at Applied Biosystems. Gene expression was analyzed by the ΔΔCt method with 18S rRNA or Gapdh as the endogenous control, and the average ΔCt of unstimulated WT controls served as the calibrator.

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.