TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thalianaviral infection

Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

doi:10.1186/s12870-014-0210-x

Table 1 Experimental conditions used in RT-qPCR based on MIQE requirements

From: TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thalianaviral infection

Experimental design
Control groups	CP#72 water treated plants/mock infected plants
Treatment groups	CP#72 methoxyfenozide treated plants/TMV-Cg infected plants
Sample
Type of sample	Arabidopsis leaves
Procesing procedure	Nitrogen liquid homogenization
Sample frozen conditions	−80°C
RNA extraction
Procedure	Acid Phenol extraction
Reagents	TRIzol
Details of Dnasa treatment	DNAsa I Amp Grade, 15 min at room temperature
Contamination assesment	<3%
Nucleic acid quantification
Instrument and method	NanoDrop instrument
Purity (A260/A280)	>1.8
RNA integrity	Analysed by agarose gel electrophoresis
Reverse transcription
Complete reaction conditions	Reaction was performed as recomendad by Invitrogen®
Amount of RNA and reaction volume	1 μg of RNA, 20 μl
Priming oligonucleotide	Oligo d(T) 20 primers (Invitrogen®) and random primers
Reverse transcriptase	MMLV® III Reverse Transcriptase
Temp and time	1 hour, 50°C
qPCR protocol
Complete reaction conditions	5 min 95°C , (30 seg 95°C, 1 min 60°C) x 40 cycles
Reaction volume and amount of cDNA	20 μl reaction, 20–200 ng de RNA
Primer, Mg and dNTPs concentration	3 mM Mg2+, 200nM primers, 0,2 mM de dNTPs
Polymerase	Taq platinum, Invitrogen
Buffer	20 mM Tris–HCl (ph 8.4), 50 mM Kcl
Manufacturer of qPCR instrument	ABI 7500, Applied Biosystems
qPCR validation
Specificity	Analysed by agarose gel and melting curve parameters on each qPCR run
Method of PCR efficiency calculation	Mean PCR efficiency per amplicon calculated by LingRegPCR program [62].
Data analysis
qPCR analysis program	LinRegPCR program
Method of Cq determination	LinRegPCR program
Outlier identification	LinRegPCR program
Justification of number and choice of reference genes	3 references genes tested (EF1α, UBQ5, GAPC1) for stability using three stability algorithms. UBQ5 was selected as the reference gene.
Description of normalization methods	Pfaffl’s mathematical model [63].
Number of technical replicates	3
Statistical method	Permutation test
Software	Fg Satistics
Repeatability (intraassay variation) C_q SD error	0.8

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