Total RNA was extracted from sorted DCs and HSPCs using an RNeasy Micro Kit (QIAGEN) and reverse transcribed with a SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNA was quantified through quantitative real-time polymerase chain reaction (PCR) using SYBR Green PCR mix (Applied Biosystems) on an Applied Biosystems PCR system. Thermocycler conditions included an initial hold period at 95°C for 2 minutes; this was followed by a 3-step PCR program, as follows: 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds for 40 cycles. Transcript abundance was calculated using the delta Ct method (normalization with 18S). The primer sequences are listed in Supplemental Table 5.

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

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.