Extended Data Figure 1 : Optimization and validation of gene expression analysis in isolated tissues.

From: Tissue-specific clocks in Arabidopsis show asymmetric coupling

Extended Data Figure 1

a, b, Relationship between enzyme concentration and opacity of the enzyme solutions (a) or processing time for vasculature and epidermis isolation (b). Higher concentrations of enzyme (>2%) lead to lower handling ability during tissue isolation because of its opacity (grey box). Mean ± s.d.; n = 6. c, The expression levels of the 10 reference genes in whole leaves, mesophyll and vasculature were detected under long-day (LD) and short-day (SD) conditions, and then the average expression stabilities M were calculated according to Vandesompele’s method9. We chose ACTIN2 (ACT2), TUBULIN5 (TUB5), POLYUBIQUITIN10 (UBQ10), ELONGATION FACTOR1α (EF1α), ASCORBATE PEROXIDASE 3 (APX3)33 and ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2)33 as commonly used reference genes, and we also chose ARABIDOPSIS TRITHORAX 3 (ATX3), REGULATORY PARTICLE TRIPLE-A 1A (RPT1a), THIOREDOXIN 3 (TRX3) and ASPARTIC PROTEINASE A1 (APA1) based on microarray analysis19. d, The expression of tissue-specific marker genes was detected in vasculature from plants grown under long-day conditions for 10 days. SULTR2;1 was used as a marker of phloem companion cells. WUSCHEL RELATED HOMEOBOX 4 (WOX4) and HOMEOBOX GENE 8 (AtHB8) were used as markers of procambium/cambium. IRREGULAR XYLEM 3 (IRX3) was used as a marker of xylem. Gene expression levels were calculated relative to LHCB2.1 expression. e, Total RNA was extracted from 10 cotyledons and 10 vasculatures grown under long-day conditions for 10 days. Extracted RNA was quantified and RNA content per single cotyledon and vasculature was estimated. n = 23. f, Expression of TOC1 and CCA1 in whole leaves, mesophyll, vasculature and epidermis. Plants were grown under long-day conditions for 10 days, and whole leaves, mesophyll, vasculature and epidermis were collected and/or isolated every 4 h. g, The expression level of the stress-induced genes COLD-REGULATED 15A (COR15A), ALCOHOL DEHYDROGENASE 1 (ADH1) and RESPONSIVE TO DESSICATION 29A (RD29A) in isolated mesophyll and vasculature with or without 50 µg ml−1 of α-Amanitin (an inhibitor of RNA polymerase II). d, f, g, The geometric mean of APA1 and IPP2 was used as a control. Mean ± s.e.m.; n = 3.