Total RNA was extracted from 2.0 to 5.0 × 106 pelleted B cells using the RNeasy Mini Kit (Qiagen). Residual DNA was removed from the extracted RNA with gDNA eliminator columns (Qiagen). cDNA was synthesized from 1.0 to 2.0 μg of total RNA with the SuperScript III First-Strand Synthesis System (Invitrogen) using oligo-dT primer. Specific transcripts were measured by real-time qRT-PCR with appropriate primers as described (6). An Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) was used to measure SYBR Green (Bio-Rad Laboratories) incorporation with the following protocol: 95°C for 15 s, 40 cycles of 94°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Data acquisition was performed during a 72°C extension step. Melting curve analysis was performed from 72° to 95°C. The change in cycling threshold (ΔΔCt) method was used to analyze levels of transcripts. Data were normalized to the expression level of β-ACTIN/β-Actin except noted otherwise (e.g., normalized to the Gapdh expression level).

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.