Gene expression analysis 41 

2.2.2.8.1.Generation and application of macroarrays

Three Oenothera species Oe. elata subsp. hookeri strain johansen (AA-I), Oe. grandiflora

strain tuscaloosa (BB-III) and Oe. argillicola strain douthat 1 (CC-V), from which fertile plastome-genome hybrids and cybrids can be produced were chosen to compare individual expression profiles. The universal vector primers M13forward and M13reverse were used to amplify PCR products of a subset of 187 selected cDNAs known or predicted to encode chloroplast proteins. All PCR products were subjected to agarose gel electrophoresis to confirm sizes and amplification quality. Once checked, each individual amplicon was adjusted to three different concentrations of 3.5, 14 and 56 ng/µl, respectively. Each dilution was spotted in duplicate onto a 7.8 x 11.9 cm positively charged Hybond N+ nylon membrane (Amersham Biosciences, New York, NJ) by 20-fold repetition to the same points using robotics equipped with a 0.4 mm 96-pin gridder (BioRobotics, Cambridge, UK). As a negative control pBluescript vector (Stratagene, La Jolla, CA) was also spotted onto the filters. After spotting, filters were denatured in 1.5 M NaCl, 0.5 M NaOH and neutralized in 0.5 M Tris, pH 7.2, 1 M NaCl. After drying, filters were cross-linked with 120 mJ of 302 nm UV light by a UV-Stratalinker 1800 (Stratagene, La Jolla, CA). 32P-dCTP labeled cDNA probes were synthesized from 10 µg total RNA as described above. The labeled cDNAs were incubated for 20 min at 37°C with RNase H- (Invitrogen, Karlsruhe, Germany) to remove

RNA. The labeled cDNAs were purified using MicroSpinTM G-50 columns (Amersham

Biosciences, New York, NJ). The arrays were pre-hybridized for 2 h at 60°C in 0.25 M phosphate buffer and 7% SDS. The labeled cDNAs were hybridized to filters overnight at 60°C. Filters were washed twice at 60°C in 2 x SSC, 0.1% SDS and twice in 1 x SSC and 0.1% SDS. Filters were then exposed on Fuji Film imaging plates (Fuji, Tokyo, Japan). The radioactive images were obtained with the FLA-3000 phosphoimager (Fuji, Tokyo, Japan). Array images were imported into the program AIDA Image Analyzer 4.0 (Raytest GmbH, Straubenhardt, Germany) and signals were deduced. For normalization, the mean value of the selected background within each sub-grid was averaged and subtracted to calculate the intensity of all spots. The duplicate signals from 3 different concentrations were averaged and the expression profiles obtained were compared to calculate the ratios with program AIDA Array Compare 4.0 (Raytest GmbH, Straubenhardt, Germany). Histograms were generated using Microsoft Excel 2003 (Microsoft, Redmond, WA).

2.2.2.8.2.Real-time PCR analysis

Real-time PCR was performed using a commercially available master mix containing Taq DNA polymerase, SYBR-Green I dye and deoxyribonucleoside triphosphates (LightCycler - FastStart DNA master SYBR-Green I, Roche Molecular Biochemicals, Mannheim, Germany). PCR products were followed by measuring SYBR Green I fluorescence. SYBR Green I dye emits a fluorescence signal at 530 nm only when bound to double-stranded DNA. Therefore, during PCR the increase in SYBR Green I fluorescence is directly proportional to the amount of double-stranded DNA generated. After addition of primers (0.5 mM), MgCl2 (4

mM), and template cDNA to the master mix, an initial denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation (95°C for 15 sec), annealing (58°C for 5 sec) and extension (72°C for 10 sec) were performed. All ramp rates were set to 20°C per sec. Detection of the fluorescent product was performed at the end of the extension period.

To prove that only the desired PCR product had been amplified, a melting curve analysis was performed after completion of PCR. For this, PCR products were denatured at 95°C, annealed at 55°C, and gradually heated to 95°C, whereas SYBR-Green I fluorescence was detected stepwise every 0.1°C. During such slow heating of the reaction mixture, melting of double- stranded DNA and a corresponding decrease of SYBR Green I fluorescence occurred. When the temperature of the reaction mixture reached the characteristic mean melting temperature of a particular DNA product (where the DNA is 50% double-stranded and 50% single- stranded), the first derivative presents a peak of a melting curve. If PCR generates only one amplicon, melting curve analysis shows only one melting peak. If primer dimers or other non- specific products are present, they cause additional melting peaks. To estimate primer-dimer formation, a control without template DNA was included in each experiment. The template quantification was determined by the crossing point using the LightCycler analysis software, as described in Wittwer et al. (1997).

2.2.2.8.2.1.Analysis of nuclear gene expression via Real-time PCR

To normalize cDNAs of Oe. elata subsp. hookeri strain johansen (AA-I), Oe. grandiflora

strain tuscaloosa (BB-III) and Oe. argillicola strain douthat 1 (CC-V), the expression of actin was measured with the primer pairs M101for/ M101rev, derived from EST cluster: S_2275-

Elip2) was measured with the primer pairs M34for/M34rev, M60for/M60rev and M75for/M75rev, respectively. Real-time PCR was performed as described above. Due to sequence dissimilarity (1.59%) among different Oenothera species primer pairs designed for the A genome did not always amplify products in BB or CC species. This was also evident by temperature shifts of melting curves indicating unspecific PCR products.

2.2.2.8.2.2.Analysis of plastid gene expression via Real-time PCR

AB-I (hjohansen·htuscaloosa Ijoh) and AB-III (hjohansen·htuscaloosa IIIlam/tusca) cDNAs were normalized to psaC using the primer pairs Ijeto and psaC5’. PsaC was chosen for normalization as no expression difference was detected in a marcoarry between the two genotpyes (Geimer and Meurer, unpublished). The expression levels of clpP and psbB were determined with the primer pairs RPL20R5M and clpP_IIP31rev for clpP, and psbBfor and psbB_IVP15rev for psbB. Real-time PCR was performed as described above.