Total RNA was isolated with DNA-free RNA isolation protocols (56 (link)). Total RNA (400 ng) was used to synthesize complementary DNA with the M-MLV Reverse Transcriptase, RNase H minus (Promega, Dübendorf, Switzerland), first-strand synthesis system. Quantitative polymerase chain reaction (PCR) analysis was performed on 10 ng of cDNA with a homemade master mix containing GoTaq polymerase (Promega) and 5× colorless GoTaq reaction buffer (Promega), 0.2 mM deoxynucleotide triphosphates, 2.5 mM MgCl2, 30 nM 6-carboxy-X-rhodamine dye, and SYBR Green in a final volume of 20 μl. Ubiquitin-conjugating enzyme (UBC21) At5g25760 (57 (link)) was used as reference gene. AOS (At5g42650) transcripts (58 (link)) were displayed relative to UBC21.
Protocol detail
Quantifying AOS Gene Expression
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Buffer
Cdna
Gene
Isolation
Mgcl2
Polymerase chain reaction
Reverse transcriptase
Rhodamine
Rnase h
Sybr green
Synthesis
Triphosphates
Ubiquitin conjugating enzyme
Corresponding Organization : University of Lausanne
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Variable analysis
independent variables
- Total RNA (400 ng)
- AOS (At5g42650) transcripts
- Ubiquitin-conjugating enzyme (UBC21) At5g25760
- Positive control: Ubiquitin-conjugating enzyme (UBC21) At5g25760 as reference gene
- Negative control: Not explicitly mentioned
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