Prime a gene kit

Total leaf RNA extracted using TRiZOL Reagent (Invitrogen) was analyzed by northern blot as previously described^{36 (link),47 (link)}. Briefly, 5 µg of denatured RNA prepared in formaldehyde containing buffer were separated by electrophoresis in a 1.5% agarose/formaldehyde gel. After blotting onto HybondN+ Nylon membranes (Amersham Biosciences), RNAs of interest were identified using ³²P-labeled DNA probes (AP24 or glucanase) synthetized by random priming with a Prime-a-Gene kit (Promega). Pre-hybridization, hybridization and washing were performed as described in^{47 (link)}.

5 µl of purified RNA samples was mixed with 3.5 µl 37% formaldehyde and 10 µl 100% formamide, 2 µl supplemented 10x MOPS buffer (400 mM MOPS buffer, 100 mM NaOAc, 10 mM Na₂EDTA). Sample mixture was incubated at 55°C for 15 min and 4 µl of loading dye (1 mM EDTA, 0.25% BromoPhenolBlue, 50% Glycerol) was added and run on an 1.5% agarose gel at 120 V in 1× MOPS buffer. ssRNA ladder (NEB) was used as size marker for all northern blot experiments.
RNA was transferred to pre-wet Nylon membrane (Hybond-N, Amersham). Blotted RNA was crosslinked to the membrane with either a UV crosslinker or by baking at 80°C for >1 h. The membrane was hybridised in hybridisation buffer (0.4 M Na₂HPO₄, 6% SDS and 1 mM EDTA) for 1 h at 57°C with RNA probes prepared using the Prime-a-Gene kit (Promega) (for list of primers used to generate RNA probes, see Table S2), washed twice with wash buffer 1 (40 mM Na₂HPO₄, 5% SDS and 1 mM EDTA) and twice with wash buffer 2 (40 mM Na₂HPO₄, 5% SDS and 1 mM EDTA). RNA was detected overnight in PhosphorImager (Molecular Dynamics).

Southern blot was performed as previously described^{47 (link)}. Briefly, 3 µg of total leaf DNA digested with NcoI (New England Biolabs, Beverly, MA, USA) were electrophoresed in 0.8% agarose gel, and blotted onto HybondN+ Nylon membrane (Amersham Biosciences, Uppsala, Sweden). The membrane was hybridized with a ³²P-labeled trnI/A DNA probe, generated by random priming with a Prime-a-Gene kit (Promega, Madison, WI, USA). Pre-hybridization and hybridization were carried out following published protocols^{47 (link),77} . The blot was analyzed after exposure to a storage phosphor screen, using a Storm 840 PhosphorImager system (Amersham Biosciences).

Total DNA was extracted from leaves as described by Dellaporta et al. (1984) (link). The DNA (2.5 μg) was digested overnight with NcoI enzyme (New England Biolabs, USA), electrophoresed in 0.8% agarose gels and blotted onto Hybond-N+ Nylon membranes (Amersham Biosciences, USA). Specific DNA sequences were detected by hybridization with α-³²P-labeled trnI/A DNA probe. The probe was generated by random priming with a Prime-a-Gene kit (Promega, USA). Pre-hybridization and hybridization were carried out at 65°C in Church’s hybridization solution (Church and Gilbert, 1984 (link)) for 2 and 16 h, respectively. Membranes were washed twice with gentle shaking for 30 min in 0.2X SSC, 0.1% SDS at 65°C. The blot was exposed to a storage phosphor screen, which was analyzed in a Storm 840 PhosphorImager system (Amersham).

Total RNA was isolated from transfected cells using Tri reagent (MRC) according to the manufacturer's protocol. The RNA was separated on a denaturing 1% agarose gel in MOPS buffer. The gel was stained with ethidium bromide to visualize total RNA (mostly comprising of rRNA) as a loading control. The RNA was transferred overnight to a nitrocellulose membrane. Subsequently, the membrane was UV crosslinked and the RNA hybridized to a ³²P-labeled probe overnight in Church buffer 65°C. The membrane was imaged using a Typhoon phosphoimager system. The probe was generated first restricting the plasmid using HindIII and XhoI (NEB), gel purifying the restricted fragment and using Prime a gene kit (Promega) in the presence of α³²P-dATP.

Total DNA was extracted from leaves harvested from 4-week-old in vitro grown plants using a modified CTAB (cetyl trimethylammonium bromide) protocol [26 (link)]. The same leaf samples were used for Western blots and reverse transcription-quantitative PCR (RT-qPCR) analysis. Ten micrograms of total DNA was restriction digested with 20 U of HindIII (Thermo Fisher) in an overnight reaction. The digested DNA was separated on a 0.8% agarose-TAE gel and transferred overnight onto a positively charged nylon membrane (Roti-Nylon Plus, Carl Roth). A 700-bp probe against the hptII gene was PCR amplified from the binary vector and labeled with [α-32P] dCTP using the Prime-A-Gene kit (Promega). The nylon membrane was treated with PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich) for 30 min followed by hybridization together with the radio-labeled probe. Blots were developed using a Typhoon FLA 7500 imaging system (GE Healthcare Life Sciences).

Ten micrograms of total RNA extracted from N. benthamiana leaves were dissolved in loading buffer (1× HEPES [20-mM HEPES, 1-mM EDTA, and 17-mM KOH, pH 7.8], 45% formamide, 16% formaldehyde, 16% ethidium bromide, and bromophenol blue) and denatured by incubation for 5 min at 95°C. RNA was run in a 1% agarose gel prepared with 16% formaldehyde 1× HEPES buffer for 3 h and transferred onto a nylon membrane (Amersham Hybond-NX, GE Healthcare Life Sciences) by capillary flow in the presence of 20× SSC buffer (3-M NaCl and 300-mM sodium citrate, pH 7.0). After UV-crosslinking (Stratalinker UV 1800, Stratagene), membranes were incubated in PerfectHyb Plus Hybridization buffer (Sigma-Aldrich) at 42°C for 1 h. A ³²P-radio-labeled probe was synthesized using the Prime-a-Gene kit (Promega, Madison, WI, USA) with an HA-NOST PCR fragment as template (see Supplemental Data Set 11 for primer sequences), and hybridized to membranes overnight at 42°C with gentle rotation in the same buffer. After hybridization, membranes were washed with 2× SSC, 2% SDS at 42°C (three washes of 10 min). Signal was detected by exposure to a phosphoimager screen (TyphoonTM FLA 7000, GE Healthcare Life Science).