Quantitative RNA Analysis by RT-qPCR and Northern Blot

Flash frozen tissue was pulverized using a Cellcrusher tissue pulverizer. RNA was then extracted using the RNeasy Plus Universal Mini Kit (Qiagen) or TRIzol (Qiagen) method and quantified using a NanoDrop spectrophotometer. For RT-qPCR, 1 or 2 µg of RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen) or Maxima Reverse Transcriptase (Invitrogen). The cDNA reaction was diluted fivefold in ultrapure water for use in RT-qPCR. RT-qPCR was performed using SYBR Select Master Mix for CFX (Applied Biosystems). The BioRad CFX96 real time PCR machine was used to carry out real time PCR and results were analyzed using the delta-delta CT method. For northern analysis, poly(A)⁺ RNA was extracted from total RNA using NucleoTrap mRNA kit (Machery-Nagel). Northern blot analysis was performed as previously described (Miura et al. 2013 (link)). Briefly, poly(A)⁺ RNA samples (2 µg) were denatured in glyoxal and run in BPTE gels prior to downward transfer followed by northern blotting using 32-P dCTP labeled DNA probes (sequences of primers used to generate probes are found in Supplemental Table 1). Blots were exposed overnight until desired intensity of signals was detected using Typhoon FLA7000 phosphoimager (GE).