Real-time RT-PCR analysis
Total RNA was extracted from LL29 cells using an RNeasy kit (Qiagen,
Hilden, Germany) according to the manufacturer’s protocol. Using a
PrimeScript™ II 1st strand cDNA Synthesis kit, samples
were reverse-transcribed and THUNDERBIRD® SYBR qPCR Mix, Bio-Rad’s
CFX96™ Real-time system, and CFX Manager™ software (Hercules, CA) were
used for real-time RT-PCR experiments. Electrophoretic analysis of
reaction products was done to confirm specificity.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as an
internal standard. Primers were designed using either Primer3 or
Primer-BLAST. Primer sequences will be provided upon request.