RNA was extracted either from the top 2 cm of stem tissue at growth stage 6.0 or from whole stems at growth stage 9.0 using the RNeasy Kit (Qiagen). cDNA was synthesized using SuperScript IV Reverse Transcriptase (Invitrogen). Quantitative polymerase chain reaction analysis was conducted to measure the transcript levels of FLA11, FLA12, primary wall cellulose synthesis genes CESA1, CESA3, and CESA6, SCW cellulose synthesis genes CESA4, CESA7, and CESA8, xylan backbone synthesis genes IRX9, IRX10, and IRX14, lignin synthesis genes PAL1, PAL2, C4H, 4CL1, REF8, HCT, CCoAOMT1, CCR1, F5H1, COMT1, and CAD5, and SCW TFs NST1 and NST3, as outlined in Table S1 . Transcript levels of each gene were assessed using a relative quantitative method (Livak & Schmittgen, 2001 (link)), with two or three biological replicates from two or three independent transformed lines, and three technical replicates in a Quant Studio 5 Real‐Time System with 384 wells (Thermo Fisher, Waltham, MA, USA) using PowerUp SYBR Green Master Mix (2X) Universal (A25742; Thermo Fisher) in 10 µl reactions. Transcript levels were normalized against housekeeping gene ACT2.
Powerup sybr green master mix 2x universal
Manufactured by Thermo Fisher Scientific
Sourced in United States
PowerUp SYBR Green Master Mix (2X) Universal is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I dye, DNA polymerase, dNTPs, and necessary buffer components.
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2 protocols using powerup sybr green master mix 2x universal
1
Transcriptional Analysis of Cell Wall Genes
2
Quantifying FLA11 Transcript Levels
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RNA was extracted from the stem tissue at growth stage 6.0 using the Spectrum Plant Total RNA Kit (Sigma #STRN250). Complementary DNA was synthesized using SuperScript IV Reverse Transcriptase (Invitrogen). RT-qPCR was conducted to measure the transcript levels of FLA11 using a relative quantitative method (Livak and Schmittgen 2001 (link)), with 2 or 3 biological replicates from 2 or 3 independent transformed lines, and 3 technical replicates in a QUANTSTUDIO 5 Real-Time System with 384 wells (Thermo Fisher, Waltham, MA, USA) using PowerUp SYBR Green Master Mix (2X) Universal (A25742; Thermo Fisher) in 10 μL reactions. Transcript levels were normalized against the housekeeping gene ACT2. RT-qPCR primers were as used in Ma et al. (2022) (link) and can be found in Supplemental Table S5 .
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