Bioanalyzer 2100 rna nano chip assays

Total RNA was isolated from needles of plants grown for six months using PureLink® Plant RNA Reagent (ThermoFisher, Waltham, MA, USA) using approximately 100 mg tissue according to manufacturer’s instructions. RNA integrity and concentration was measured using Bioanalyzer 2100 RNA Nano chip assays (Agilent, Santa Clara, CA, USA) following the manufacturer’s protocol. Equal RNA amounts were used for cDNA synthesis with the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). qRT-PCR reactions were performed on a Bio-Rad CFX96 Real-time system using the SsoFast kit (Bio-Rad, Hercules, CA, USA) in triplicate. Relative transcript abundance was calculated using efficiency corrected ΔC_T and ΔΔC_T values based on ELF-1α as the reference gene. Target-specific oligonucleotides were as follows: ELF-1α-f—5′-CCCTTCCTCACTCCAACTGCATA; ELF-1α-r—5′-TCGGCGGTGGCAGAGTTTACATTA; or PgβGLU1-f—5′-TTGGATCCTCTGAAGGT GT; PgβGLU1-r—5′-TCCCTCCCTTATGGCTTC. Target specificity was confirmed by sequence verification of representative amplicons.

Total RNA was isolated as previously described using approximately 100 mg tissue [75 (link)]. RNA integrity and concentration was measured using Bioanalyzer 2100 RNA Nano chip assays (Agilent) following the manufacturer’s protocol. Equal RNA amounts were used for cDNA synthesis with the Superscript III reverse transcriptase (ThermoFisher) and oligo(dT) primers. The subsequent qPCR reaction was performed on a Bio-Rad CFX96 Real-time system using the SsoFast kit (www.bio-rad.com) and target-specific oligonucleotides (Additional file 9: Table S2). Relative transcript abundance was calculated using efficiency corrected ΔCT using the amplification efficiency values (E-values) generated from primer efficiency calculations for each gene pair. Relative gene expression values were calculated based on M. vulgare Elongation Factor 1α as reference gene and triplicate measurements with three biological replicates. Target specificity was confirmed by sequence verification of representative amplicons.