Improm 2 reverse transcriptase

Total RNA isolation from Arabidopsis plants for use in RT-PCR analysis was described previously^{38 (link)}. Plant tissues were harvested, and the RNA was isolated using TRIzol Reagent (Invitrogen, USA). After being treated with DNase I (Promega), 2 μg of total RNA was subjected to reverse transcription reaction using the IMPROM II reverse transcriptase (Invitrogen, USA) at 25 °C for 5 min, 42 °C for 1 h and 70 °C for 15 min. The resulting cDNA was used for PCR amplification with the gene-specific primers. The Arabidopsis ACTIN8 (ACT8) gene was used as internal controls. Sequences of the primers used are listed in Supplementary Table S1.

Mouse tissues were pulverized in liquid N₂ with mortar and pestle, and total RNA was extracted with Trizol (Invitrogen). One microgram of total RNA was reverse-transcribed with ImProm-II Reverse Transcriptase (Invitrogen). For radioactive RT–PCR, the human-specific primer pair E4-33to55-F and E8-15to36-R was used for amplifying human SMN2 transcripts in RNA samples from mouse tissues, as described (25 (link)); all PCR products were labeled with α-³²P-dCTP and analyzed by 6% native polyacrylamide gels, followed by phosphorimage analysis. The extent of exon 7 inclusion was calculated as described (25 (link)), and the signal intensity of each cDNA band was normalized according to its G+C content.