Biology Reference
In-Depth Information
Quantitative Real-Time PCR
We germinated seeds of the wild type and wrky2 mutants on MS medium with or
without 1.5
µ
M ABA for 4 or 7 days, and germinated the seeds of abi5-1, abi3-1,
aba2-3, aba3-1 mutants and the wild type (Col, Ws and Ler) on MS medium
with or without 1.5
µ
M ABA for 4 days. Harvest samples were froze immediately
in liquid nitrogen, and stored at 80°C. RNA was extracted from these samples
using An RNeasy Plant Mini kit (QIAgen, Valencia, CA), and DNA was removed
via an on-column DNase treatment. For real-time PCR, the First Strand cDNA
Synthesis kit (Roche, Diagnostics, Mannheim, Germany) was used to make
cDNA from 1
µ
g of RNA in a 20
µ
L reaction volume. Each cDNA sample was
diluted 1:20 in water, and 2
µ
L of this dilution was used as template for qPCR.
Half-reactions (10
µ
L each) were performed with the Lightcycler FastStart DNA
Master SYBR Green I kit (Roche, Mannheim, Germany) on a Roche LightCy-
cler real-time PCR machine, according to the manufacturer's instructions. ACT2
(AT3G18780) was used as a control in qPCR. Gene-specific primers for detect-
ing transcripts of ACT2, WRKY2, ABI5, ABI3, Em1 and Em6 are listed in Table
2. Gene-specific primers of MYB33 and MYB101 are as described by Allen et
al. (2007) [53]. The qPCR reactions (10
µ
L each) for these genes contained the
following: 1
µ
L SYBR Green I reaction mix, 3 mM MgCl
2
, 0.5
µ
M forward and
reverse primers and 2
µ
L cDNA. The annealing temperature was 52°C in all cases.
A no-template control was routinely included to confirm the absence of DNA or
RNA contamination. The mean value of four replicates was normalized using the
ACT2 gene as the control. Standard curves were generated using linearized plas-
mid DNA for each gene of interest. A second set of experiments was conducted
on an independent set of tissue as a control.
Table 2.
List of quantitative RT-PCR primer sequences
