Ambion trizol rna isolation reagent

For analyzing FLC expression we used plants grown simultaneously with the plants of the NIL drought experiment described above. All above ground tissues were harvested for each plant 21 days after sowing. Tissue from two plants was pooled for each of three biological replicates. RNA was extracted using Trizol (Ambion® TRIzol® RNA Isolation Reagent, Life Technologies) and transcribed into cDNA using Super Script® II Reverse Transcriptase (Invitrogen). Quantitative RT-PCR was performed on a CFX384 Touch™ Real-Time PCR Detection System (Biorad) using SYBR Green dye (iQ™ SYBR® Green Supermix, Biorad) and the following FLC specific primers: F-primer: 5′-CCGAACTCATGTTGAAGCTTGTTGAG-3′, R-primer: 5′-CGGAGATTTGTCCAGCAGGTG-3′. Expression values were determined using the standard curve method and normalized to the expression of PP2A (F-primer: 5′-TAACGTGGCCAAAATGATGC-3′, R-primer: 5′- GTTCTCCACAACCGCTTGGT-3′). Normalized expression was averaged for three biological replicates each analyzed in three technical replicates.

For expression analysis of transgenics, seedlings (leaves plus shoot) grown in an environmental chamber at long day conditions (16 h light) were harvested 10 days after sowing. Material of 8 to 10 seedlings was pooled for each of three biological replicates. RNA was extracted using Trizol (Ambion® TRIzol® RNA Isolation Reagent, Life Technologies) and transcribed into cDNA using Super Script® II Reverse Transcriptase (Invitrogen). Quantitative RT-PCR was performed on a CFX384 Touch™ Real-Time PCR Detection System using SYBR Green dye (iQ™ SYBR® Green Supermix, Biorad) using the following gene-specific primers: FLC-fwd: 5′-CCGAACTCATGTTGAAGCTTGTTGAG-3′, FLC-rev: 5′- CGGAGATTTGTCCAGCAGGTG-3′, FRI-fwd: 5′- TGCCTGATCGTGGTAAAGGGAAG-3′ and FRI-rev: 5′- AGCACCGGCAATCTCATTCGAAC-3′. Expression values were determined using the standard curve method and normalized to the expression of PP2A (PP2A-fwd: 5′-TAACGTGGCCAAAATGATGC-3′, PP2A-rev: 5′- GTTCTCCACAACCGCTTGGT-3′). Normalized expression was averaged for three biological replicates each analyzed in two or three technical replicates.