Sybr green

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) experiments were performed with 5 μl of a 1:50 dilution of first-strand cDNA and SYBR Green, according to the manufacturer’s instructions (Eurogentec SA, Seraing, Belgium). Gene-specific oligonucleotides (S1 Table) were designed with Primer3 software (http://frodo.wi.mit.edu), and their specificity was checked by analyzing dissociation curves after each run. Genes encoding a mitochondrial NADH-ubiquinone oxidoreductase subunit (AT5G11770) and a mitochondrial inner membrane translocase (AT5G62050) were selected as constitutive internal controls [46 (link)]. For microarray validation, RNA was isolated from non-inoculated roots (NI), and from roots 2.5 hours after inoculation (hai), 6 hai, 10.5 hai and 30 hai with P. parasitica. Two biological replicates of the entire experiment were performed, each as a technical triplicate. For each time point, six results were analyzed. Gene expression was quantified and normalized with respect to constitutively expressed internal controls [18 ].
For VQ29 and hormonal pathway expression analysis for mutant validation, RNA was isolated from non-inoculated roots (NI), and from roots 6 hours after inoculation. Three biological replicates of each time points were performed, each as a technical triplicate. Analysis was performed as above.

Total RNA was extracted using Tissue/cell RNA Mini kit (Invitrogen) according to the manufacturer's protocol. The cDNAs were generated from 1 μg of total RNA by superscript II RNase H-reverse transcriptase with Oligo (dT) primer. Quantitative PCR reaction was achieved using commercial kit containing SYBR Green (Eurogentec, Seraing, Belgium). The primer pairs (Invitrogen) were as follows: AQP4 (MN-012825, 5′-agaaccaaggcgtaaaccg-3′ and 5′-tccctggaaatgactgagaaa-3′, 256 bp), DG (NM_053697.1, 5′-tagcgtccctgacatccg-3′ and 5′-gaatcagttgaaggcgttgc-3′, 516 bp), and β-actin (NM_031144, 5′-ctgccgcatcctcttcctc-3′ and 5′-ctcctgcttgctgatccacat-3′, 398 bp). The cycling program was 5 min at 95°C and then 40 cycles, each consisting of 15 s at 95°C and 30 s at 60°C. Changes in each mRNA expression were examined with ABI7000 Sequence Detection System (Perkin Elmer, USA). A standard curve was used to extrapolate the copy number of target cDNA in rat brain.

RNA was isolated from BUMPT cells using the TRIzol method (Life Technologies, Darmstadt, Germany) according to the manufacturer’s instructions. RNA quality and integrity were ensured by the A260/280 ratio and agarose gel electrophoresis, respectively. One microgram of RNA was used for the reverse transcription reaction. DNase 1 (1 U/1 µg RNA) treatment was performed for 30 min at 37 °C. Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qRT–PCR was performed using an Applied Biosystems ViiA 7 Real-time System (Thermo Fisher Scientific, Dreieich, Germany), SYBR Green (Takyon™, Eurogentec, Cologne, Germany), and primers (Supplemental File S2). For quantitative analysis, the results were normalized to 18S RNA. The ΔΔCt method was used to analyze relative gene expression [22 (link)].

Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2 µg RNA were transcribed into cDNA using the SuperScript First Strand Synthesis Kit (Invitrogen). Transcript levels of Puma and L32 were quantified by quantitative RT-PCR on an CFX96TM Real Time System thermocycler (Bio-Rad) with SYBR Green (Eurogentec, Lüttich, Belgium). The following primers were used: mPuma: 5′-GCCCAGCAGCACTTAGAGTC-3′ and 5′-GGTGTCGATGCTGCTCTTCT-3′; mL32: 5′-TTAAGCGAAACTGGCGGAAAC-3′ and 5′-TTGTTGGTCCCATAACCGATG-3′.

RNA extraction, cDNA synthesis and quantitative real-time RT-PCR (qRT-PCR) were performed as previously reported [30 (link)] using SYBR green (Eurogentec) for detection. The data were expressed relative to the housekeeping gene HPRT and calculated using the 2^-∆∆Ct method. The primer sequences used were: HPRT (F) 5’-ATTATGCTGAGGATTTGGAAAGGG-3′ and (R) 5’-GCCTCCCATCTCCTTCATCAC-3′; CD133 (F) 5’-CAATCTCCCTGTTGGTGATTTG-3′ and (R) 5’-ATCACCAGGTAAGAACCCGGA-3′; VEGF (F) 5’-CCAAGTGGTCCCAGGCTGCA-3′ and (R) 5’-TGGATGGCAGTAGCTGCGCT-3′; HIF1A (F) 5’-CCTCTGTGATGAGGCTTACCATC-3′ and (R) 5’-CATCTGTGCTTTCATGTCATCTTC-3′, HIF2A (F) 5’-CCACCAGCTTCACTCTCTCC-3′ and (R) 5’-TCAGAAAAGGCCACTGCTT-3′.