RNA isolation and transcript analysis for CAB and RBCS transcripts.
After 3 days of stratification at 4°C, Col-0, mid-2 and cop1-4 seedlings of each biological replicate
were grown for 4 days at 21°C in the dark in three sectors of a shared MS plates lacking sucrose. RNA
was prepared using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com/). RNA quality was
tested by photometric analysis (A260/A280 ratio between 1.9 and 2.1) and gave two distinct bands in
formaldehyd agarose gel electrophoresis. Test-PCR with 35 cycles using EF1aA4 primers (Kirik et al.,
2007) resulted in a band of 709 bp (cDNA) that indicated that the samples were devoid of DNA, while
a sample of genomic DNA gave a clear band at 809 bp (genomic DNA). RNA was subjected to
digestion with DNase I (Thermo Scientific, http://www.thermoscientific.com/fermentas) and
subsequent reverse transcription was done using the Superscript III First strand System for RT-PCR
including RNaseH treatment (Invitrogen, www.invitrogen.com). Transcript levels were determined by
quantitative
PCR
using
an
Applied
Biosystems
7300
real-time
PCR
system
(http://www.appliedbiosystems.com/) analyzing triplicates of 1 µl cDNA in a 25 µl reaction
containing
POWER
SYBR
Green
PCR
Master
Mix
(Applied
Biosystems,
www.appliedbiosystems.com) and gene-specific primers for UBQ10, CAB and RBCS (Table S5).
Dissociation curves were analyzed. Baseline and threshold were set manually according to the
manufacturer`s instructions. Two biological replicates were used and each was analyzed in duplicate.
The results were analyzed by the Ct method using UBQ10 for normalization (manufacturer`s
instructions and Bookout and Mangelsdorf, 2003). Statistical analysis was performed by applying a
one tailed Welch-test (Ruxton, 2006).
Supplemental literature
Bookout, A. L., Mangelsdorf, D. J. (2003) Quantitative real-time PCR protocol for analysis
of nuclear receptor signaling pathways. Nucl Recept Signal, 1, e012.