MIAME checklist
Experiment design
Goal of the experiments: Analysis of expression changes in Arabidopis plants after challenge with two insect herbivores of different diet breadth: the specialist Pieris rapae and the generalist Spodoptera littoralis. Analysis of expression changes in Arabidopsis plants after dehydration.
Description of the experiments: The model plant used is Arabidopsis thaliana. The first type of experiment is measuring relative changes in gene expression after 4-5 h challenge with P. rapae and S. littoralis in wild type and different signaling mutant plants using a dedicated cDNA microarray. The mutants used in this study were coi1-1 (impaired in the perception of jasmonic acid), sid2-1 (does not accumulate salicylic acid after pathogen inoculation), npr1-1 (impaired in the SA signaling pathway), ein2-1 (ethylene insensitive), and aba2-1 and aba3-1 (accumulate little abscisic acid). Since all of these mutants are in the Col-0 background, this genotype was used as a reference. The second type of experiment is measuring gene expression after 8 days of feeding by S. littoralis larvae. Only the mutant aba2-1 and Col-0 were used for this set of experiments. The third type of experiment is dehydration (Col-0 plants were left without water for 5-6 days). To estimate biological and technical variation in gene expression, a series of 4 microarrays were done comparing untreated Col-0 plants to untreated Col-0 plants.
Keywords: genotype comparison, treatement = dehydration or insect feeding
Experimental factors: genotype, treatment, time
Experiment design:
Experiment / Genotype / Cy3 sample / Cy5 sample / Number of replicatesA / Col-0 / control / treated (4-5h P. rapae) / 8
B / coi1-1 / control / treated (4-5h P. rapae) / 4
C / ein2-1 / control / treated (4-5h P. rapae) / 3
D / sid2-1 / control / treated (4-5h P. rapae) / 4
E / aba2-1 / control / treated (4-5h P. rapae) / 4
F / aba3-1 / control / treated (4-5h P. rapae) / 4
G / npr1-1-1 / control / treated (4-5h P. rapae) / 4
H / Col-0 / control / treated (4-5h S. littoralis) / 6
I / coi1-1 / control / treated (4-5h S. littoralis) / 4
J / ein2-1 / control / treated (4-5h S. littoralis) / 4
K / sid2-1 / control / treated (4-5h S. littoralis) / 5
L / aba2-1 / control / treated (4-5h S. littoralis) / 4
M / aba3-1 / control / treated (4-5h S. littoralis) / 4
N / npr1-1 / control / treated (4-5h S. littoralis) / 3
O / Col-0 / control / treated (8 days S. littoralis) / 5
P / aba2-1 / control / treated (8 days S. littoralis) / 2
Q / Col-0 / control / treated (dehydration) / 3
R / Col-0 / control / control / 4
Quality controls: independent biological replicates were carried-out
- For P. rapae treatment: 8 biological replicates for Col-0, 4 biological replicates for coi1-1, aba2-1, aba3-1, sid2-1, npr1-1, 3 biological replicates for ein2
- For S. littoralis 4-5 h treatment: 6 biological replicates for Col-0, 5 biological replicates for sid2-1, 4 biological replicates for coi1-1, aba2-1, aba3-1, npr1-1, ein2-
- For S. littoralis 8 days treatment: 5 biological replicates for Col-0, 2 biological replicates for aba2-1
- For dehydration treatment: 3 biological replicates
Links to the publication and supplemental websites: http://www.unil.ch/dbmv/page13790_en.html
Samples used, extract preparation and labelling
The origin of each biological sample and its characteristics:
6 week-old Arabidopisis rosette leaves grown under 10-h light / 14-h dark photoperiod, from wild-type or mutant plants.
Manipulations of biological samples:
4-5 hours feeding by 4th and 5th instar larvae of Pieris rapae, starting at 10 am.
4-5 hours feeding by 4th and 5th instar larvae of Spodoptera littoralis, starting at 10 am.
8 days feeding by 1st to 3rd instar larvae of Spodoptera littoralis
5-6 days dehydration (plants were not watered).
Experimental factor value:
Sample / Experiment / Replicate / Dye / Genotype / Treatement / Time1 / A / 1 / Cy3 / Col-0 / control / 5h
2 / A / 1 / Cy5 / Col-0 / P. rapae feeding / 5h
3 / A / 2 / Cy3 / Col-0 / control / 5h
4 / A / 2 / Cy5 / Col-0 / P. rapae feeding / 5h
5 / A / 3 / Cy3 / Col-0 / control / 5h
6 / A / 3 / Cy5 / Col-0 / P. rapae feeding / 5h
7 / A / 4 / Cy3 / Col-0 / control / 5h
8 / A / 4 / Cy5 / Col-0 / P. rapae feeding / 5h
9 / A / 5 / Cy3 / Col-0 / control / 5h
10 / A / 5 / Cy5 / Col-0 / P. rapae feeding / 5h
11 / A / 6 / Cy3 / Col-0 / control / 5h
12 / A / 6 / Cy5 / Col-0 / P. rapae feeding / 5h
13 / A / 7 / Cy3 / Col-0 / control / 5h
14 / A / 7 / Cy5 / Col-0 / P. rapae feeding / 5h
15 / A / 8 / Cy3 / Col-0 / control / 5h
16 / A / 8 / Cy5 / Col-0 / P. rapae feeding / 5h
17 / B / 1 / Cy3 / coi1-1 / control / 5h
18 / B / 1 / Cy5 / coi1-1 / P. rapae feeding / 5h
19 / B / 2 / Cy3 / coi1-1 / control / 5h
20 / B / 2 / Cy5 / coi1-1 / P. rapae feeding / 5h
21 / B / 3 / Cy3 / coi1-1 / control / 5h
22 / B / 3 / Cy5 / coi1-1 / P. rapae feeding / 5h
23 / B / 4 / Cy3 / coi1-1 / control / 5h
24 / B / 4 / Cy5 / coi1-1 / P. rapae feeding / 5h
25 / C / 1 / Cy3 / ein2-1 / control / 5h
26 / C / 1 / Cy5 / ein2-1 / P. rapae feeding / 5h
27 / C / 2 / Cy3 / ein2-1 / control / 5h
28 / C / 2 / Cy5 / ein2-1 / P. rapae feeding / 5h
29 / C / 3 / Cy3 / ein2-1 / control / 5h
30 / C / 3 / Cy5 / ein2-1 / P. rapae feeding / 5h
31 / D / 1 / Cy3 / sid2-1 / control / 5h
32 / D / 1 / Cy5 / sid2-1 / P. rapae feeding / 5h
33 / D / 2 / Cy3 / sid2-1 / control / 5h
34 / D / 2 / Cy5 / sid2-1 / P. rapae feeding / 5h
35 / D / 3 / Cy3 / sid2-1 / control / 5h
36 / D / 3 / Cy5 / sid2-1 / P. rapae feeding / 5h
37 / D / 4 / Cy3 / sid2-1 / control / 5h
38 / D / 4 / Cy5 / sid2-1 / P. rapae feeding / 5h
39 / E / 1 / Cy3 / aba2-1 / control / 5h
40 / E / 1 / Cy5 / aba2-1 / P. rapae feeding / 5h
41 / E / 2 / Cy3 / aba2-1 / control / 5h
42 / E / 2 / Cy5 / aba2-1 / P. rapae feeding / 5h
43 / E / 3 / Cy3 / aba2-1 / control / 5h
44 / E / 3 / Cy5 / aba2-1 / P. rapae feeding / 5h
45 / E / 4 / Cy3 / aba2-1 / control / 5h
46 / E / 4 / Cy5 / aba2-1 / P. rapae feeding / 5h
47 / F / 1 / Cy3 / aba3-1 / control / 5h
48 / F / 1 / Cy5 / aba3-1 / P. rapae feeding / 5h
49 / F / 2 / Cy3 / aba3-1 / control / 5h
50 / F / 2 / Cy5 / aba3-1 / P. rapae feeding / 5h
51 / F / 3 / Cy3 / aba3-1 / control / 5h
52 / F / 3 / Cy5 / aba3-1 / P. rapae feeding / 5h
53 / F / 4 / Cy3 / aba3-1 / control / 5h
54 / F / 4 / Cy5 / aba3-1 / P. rapae feeding / 5h
55 / G / 1 / Cy3 / npr1-1 / control / 5h
56 / G / 1 / Cy5 / npr1-1 / P. rapae feeding / 5h
57 / G / 2 / Cy3 / npr1-1 / control / 5h
58 / G / 2 / Cy5 / npr1-1 / P. rapae feeding / 5h
59 / G / 3 / Cy3 / npr1-1 / control / 5h
60 / G / 3 / Cy5 / npr1-1 / P. rapae feeding / 5h
61 / G / 4 / Cy3 / npr1-1 / control / 5h
62 / G / 4 / Cy5 / npr1-1 / P. rapae feeding / 5h
63 / H / 1 / Cy3 / Col-0 / control / 5h
64 / H / 1 / Cy5 / Col-0 / S. littoralis feeding / 5h
65 / H / 2 / Cy3 / Col-0 / control / 5h
66 / H / 2 / Cy5 / Col-0 / S. littoralis feeding / 5h
67 / H / 3 / Cy3 / Col-0 / control / 5h
68 / H / 3 / Cy5 / Col-0 / S. littoralis feeding / 5h
69 / H / 4 / Cy3 / Col-0 / control / 5h
70 / H / 4 / Cy5 / Col-0 / S. littoralis feeding / 5h
71 / H / 5 / Cy3 / Col-0 / control / 5h
72 / H / 5 / Cy5 / Col-0 / S. littoralis feeding / 5h
73 / H / 6 / Cy3 / Col-0 / control / 5h
74 / H / 6 / Cy5 / Col-0 / S. littoralis feeding / 5h
75 / I / 1 / Cy3 / coi1-1 / control / 5h
76 / I / 1 / Cy5 / coi1-1 / S. littoralis feeding / 5h
77 / I / 2 / Cy3 / coi1-1 / control / 5h
78 / I / 2 / Cy5 / coi1-1 / S. littoralis feeding / 5h
79 / I / 3 / Cy3 / coi1-1 / control / 5h
80 / I / 3 / Cy5 / coi1-1 / S. littoralis feeding / 5h
81 / I / 4 / Cy3 / coi1-1 / control / 5h
82 / I / 4 / Cy5 / coi1-1 / S. littoralis feeding / 5h
83 / J / 1 / Cy3 / ein2-1 / control / 5h
84 / J / 1 / Cy5 / ein2-1 / S. littoralis feeding / 5h
85 / J / 2 / Cy3 / ein2-1 / control / 5h
86 / J / 2 / Cy5 / ein2-1 / S. littoralis feeding / 5h
87 / J / 3 / Cy3 / ein2-1 / control / 5h
88 / J / 3 / Cy5 / ein2-1 / S. littoralis feeding / 5h
89 / J / 4 / Cy3 / ein2-1 / control / 5h
90 / J / 4 / Cy5 / ein2-1 / S. littoralis feeding / 5h
91 / K / 1 / Cy3 / sid2-1 / control / 5h
92 / K / 1 / Cy5 / sid2-1 / S. littoralis feeding / 5h
93 / K / 2 / Cy3 / sid2-1 / control / 5h
94 / K / 2 / Cy5 / sid2-1 / S. littoralis feeding / 5h
95 / K / 3 / Cy3 / sid2-1 / control / 5h
96 / K / 3 / Cy5 / sid2-1 / S. littoralis feeding / 5h
97 / K / 4 / Cy3 / sid2-1 / control / 5h
98 / K / 4 / Cy5 / sid2-1 / S. littoralis feeding / 5h
99 / K / 5 / Cy3 / sid2-1 / control / 5h
100 / K / 5 / Cy5 / sid2-1 / S. littoralis feeding / 5h
101 / L / 1 / Cy3 / aba2-1 / control / 5h
102 / L / 1 / Cy5 / aba2-1 / S. littoralis feeding / 5h
103 / L / 2 / Cy3 / aba2-1 / control / 5h
104 / L / 2 / Cy5 / aba2-1 / S. littoralis feeding / 5h
105 / L / 3 / Cy3 / aba2-1 / control / 5h
106 / L / 3 / Cy5 / aba2-1 / S. littoralis feeding / 5h
107 / L / 4 / Cy3 / aba2-1 / control / 5h
108 / L / 4 / Cy5 / aba2-1 / S. littoralis feeding / 5h
109 / M / 1 / Cy3 / aba3-1 / control / 5h
110 / M / 1 / Cy5 / aba3-1 / S. littoralis feeding / 5h
111 / M / 2 / Cy3 / aba3-1 / control / 5h
112 / M / 2 / Cy5 / aba3-1 / S. littoralis feeding / 5h
113 / M / 3 / Cy3 / aba3-1 / control / 5h
114 / M / 3 / Cy5 / aba3-1 / S. littoralis feeding / 5h
115 / M / 4 / Cy3 / aba3-1 / control / 5h
116 / M / 4 / Cy5 / aba3-1 / S. littoralis feeding / 5h
117 / N / 1 / Cy3 / npr1-1 / control / 5h
118 / N / 1 / Cy5 / npr1-1 / S. littoralis feeding / 5h
119 / N / 2 / Cy3 / npr1-1 / control / 5h
120 / N / 2 / Cy5 / npr1-1 / S. littoralis feeding / 5h
121 / N / 3 / Cy3 / npr1-1 / control / 5h
122 / N / 3 / Cy5 / npr1-1 / S. littoralis feeding / 5h
123 / O / 1 / Cy3 / Col-0 / control / 8 days
124 / O / 1 / Cy5 / Col-0 / S. littoralis feeding / 8 days
125 / O / 2 / Cy3 / Col-0 / control / 8 days
126 / O / 2 / Cy5 / Col-0 / S. littoralis feeding / 8 days
127 / O / 3 / Cy3 / Col-0 / control / 8 days
128 / O / 3 / Cy5 / Col-0 / S. littoralis feeding / 8 days
129 / O / 4 / Cy3 / Col-0 / control / 8 days
130 / O / 4 / Cy5 / Col-0 / S. littoralis feeding / 8 days
131 / O / 5 / Cy3 / Col-0 / control / 8 days
132 / O / 5 / Cy5 / Col-0 / S. littoralis feeding / 8 days
133 / P / 1 / Cy3 / aba2-1 / control / 8 days
134 / P / 1 / Cy5 / aba2-1 / S. littoralis feeding / 8 days
135 / P / 2 / Cy3 / aba2-1 / control / 8 days
136 / P / 2 / Cy5 / aba2-1 / S. littoralis feeding / 8 days
137 / Q / 1 / Cy3 / Col-0 / control / 5-6 days
138 / Q / 1 / Cy5 / Col-0 / dehydration / 5-6 days
139 / Q / 2 / Cy3 / Col-0 / control / 5-6 days
140 / Q / 2 / Cy5 / Col-0 / dehydration / 5-6 days
141 / Q / 3 / Cy3 / Col-0 / control / 5-6 days
142 / Q / 3 / Cy5 / Col-0 / dehydration / 5-6 days
143 / R / 1 / Cy3 / Col-0 / control / 0h
144 / R / 1 / Cy5 / Col-0 / control / 0h
145 / R / 2 / Cy3 / Col-0 / control / 0h
146 / R / 2 / Cy5 / Col-0 / control / 0h
147 / R / 3 / Cy3 / Col-0 / control / 0h
148 / R / 3 / Cy5 / Col-0 / control / 0h
149 / R / 4 / Cy3 / Col-0 / control / 0h
150 / R / 4 / Cy5 / Col-0 / control / 0h
Technical protocols:
1. RNA Extraction
· Grind 3-6 g (about 12 plants) of Arabidopsis leaves in liquid N2 with mortar and pestle.
· Transfer to 50 ml Falcon tube.
· Add 18 ml of 1:2:1 solution (precipitation at RT)
o 1 volume of 2M Tris-HCl pH 8.2
o 2 volumes of 0.5M EDTA pH 8.0 (DEPC treated)
o 1 volume of 20% SDS
· Add 9 ml of phenol saturated with 0.1M Tris-HCl pH 8.2. Stir for minimum 15min.
· Add 9 ml chloroform. Stir for minimum 10min.
· Centrifuge 4500rpm 15min.
· Treat upper phase (aqueous) twice with 9 ml chloroform, centrifuging 4500rpm 15min.
· Transfer 15 ml of aqueous phase to 30 ml disposable polypropylene tube and add equal volume of ice-cold 6M LiCl (DEPC treated). Precipitate O/N on ice at 4°C.
· Centrifuge 9000rpm 20min 4°C.
· Resuspend into 2.5 ml H2O, add 0.25 ml 3M NaAcetate pH 5.2 and 3 volumes (8.3 ml) of EtOH. Precipitate at RT for ca 60min.
· Centrifuge 9000rpm 20min 16°C, rinse with 5ml 70% EtOH, centrifuge 9000rpm 5min, air dry pellet 15min and resuspend in 0.5 ml H2O.
· Quantify RNA using Nanodrop. Dilute 2ml in 400ml 10 mM Tris pH 8.
2. Probe labeling
Concentrate RNA using Microcon
· take 100 µg RNA in 80 µl H2O, put in a Microcon (Ultracel YM-30) column
· centrifuge 4min at 14000g
· turn column in clean tube
· centrifuge for 3 min at 1000 g
· measure concentration (0.5 µl in 300 µl 10 mM Tris pH8)
Reverse transcription
· prepare two 200 ml PCR tubes according to the recipe below:
· 50 mg of total RNA
· 2 ml oligo dT21 mer (1 mg/ml)
· enough water to achieve 13.4 ml final volume
· Heat 5 min at 70oC in PCR machine. Leave at RT for 5 min
· Prepare mix according to the following recipe
o 6 ml 5x SuperScript II Buffer
o 3 ml 0.1 M DTT
o 0.6 ml dNTPS (25 mM dATP, dGTP, dTTP; 10 mM dCTP)
o 2 ml SuperScript II Reverse Transcriptase
o 2 ml RNase Inhibitor
· add 13.6 ml mix per tube