Hybond n membrane

Total RNA was extracted from wild type and kanamycin screened 0–9897 jute seedlings of hpRNA transgenic lines (COMT & C4H) using the guanidium thiocynate extraction method. For northern blot of the transcripts 30 μg of total RNA from each plant sample was resolved on a 1.2% formaldehyde-agarose gel and transferred on to Hybond N + membranes (GE Healthcare Life Sciences, UK). The probes that spanned the entire coding sequence of the corresponding genes were prepared using [α^32P] dCTP labelled cDNA of COMT and C4H genes individually.
For northern blot of hpRNA, total RNA was isolated from non-transgenic and transgenic hpRNA (COMT and C4H) lines using TRIZOL reagent and following the manufacturer’s protocol (Invitrogen). 20 μg of each sample was resolved on a 12% denaturing urea-PAGE gel. The RNA was then blotted onto a Hybond-N + membrane (GE Healthcare Life Sciences, UK) by Semi-Dry Transfer Cell (Bio-Rad Laboratories, Inc, CA). Probes were prepared using [α^32P] dCTP labelled cDNAs that enclosed the respective hpRNAi amplicon specific sequences of COMT and C4H genes. Probes were purified in a G25 column (GE Healthcare Life Sciences, UK) according to the supplier’s protocol. Hybridization was carried out at 42 °C using a standard protocol. The membranes were subjected to autoradiography using a TYPHOON phosphor imager (GE Healthcare Life Sciences, UK).

Steady-state levels of mitochondrial transcripts were determined by Northern blot analysis, using 3 μg of total RNA. RNA samples were separated in 1% MOPS-formaldehyde agarose gels and transferred to Hybond-N+ membranes (GE Healthcare). To analyse mitochondrial tRNA steady-state levels, samples were separated in neutral 10% PAGE and transferred to Hybond-N+ membranes (GE Healthcare). Membranes were hybridised with either randomly [³²P]-labelled dsDNA probes or in vitro transcribed single-stranded RNA probes to detect mRNAs and rRNAs or with strand-specific [³²P]-end labelled oligonucleotide probes to detect tRNAs. Membranes were exposed to a PhosphorImager screen and the signal was quantified using a Typhoon 7000 FLA and the ImageQuant TL 8.1 software (GE Healthcare). Primers used to generate dsDNA probes and oligonucleotide probes have been previously described [10 (link),23 (link),36 (link)].

Southern blot analysis was performed as described previously [64 (link)]. Briefly, genomic DNA was isolated from the wild-type strain and scre4 knockout candidates using CTAB extraction buffer (100 mM Tris-Cl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 3% CTAB, 0.2% β-mercaptoethanol) and was then digested with Pst I overnight. The digested DNA was separated in 1% agarose gel and was then blotted on the Hybond^TM-N⁺ membrane (GE Healthcare, Amersham, UK). The membrane was hybridized with the digoxin-labeled probe using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

RNA samples were mixed with an equal volume of 2× formamide loading buffer (93% formamide, 0.1× Tris/Borate/EDTA (TBE), 30 mM EDTA, 0.03% bromophenol blue, and 0.03% xylene cyanol), heated at 95 °C for 5 min and then electrophoresed on a 4% polyacrylamide/7 M urea/1× TBE denaturing gel. Then, the RNA was transferred onto a Hybond^TM-N⁺ membrane (GE Healthcare) in 1× BE at 1 A for 1–2 h and cross-linked to the membrane under UV light at 254 nm and 1200 × 100 μJ/cm². The membrane was pre-hybridized in Church buffer (0.5 M Na₂HPO₄-H₃PO₄ buffer pH 7.2, 1 mM EDTA, 7% SDS, and 1% BSA) at 35 °C for 30 min and then in Church buffer with 5′-end-labeled oligo probes^{36 (link)} (Table S3) at 35 °C overnight. Subsequently, the membrane was washed once with 2× SSC and 0.1% SDS at 50 °C for 20 min and then twice with 0.1× SSC and 0.1% SDS at 50 °C for 20 min each time. The signals on the membrane were detected with a Typhoon FLA 9500 (GE Healthcare). Probes specific to GFP mRNA³⁷ were labeled with terminal deoxynucleotidyl transferase (Thermo Scientific).

HBV cccDNA was isolated by the Hirt procedure and detected by southern blot as described previously [26 (link)]. Briefly, the Hirt DNA samples were heated at 85 °C for 5 min to denature rcDNA into single-stranded DNA, followed by plasmid-safe ATP-dependent DNase treatment (Epicentre, Illumina Inc., Madison, WI, USA) at 37 °C for 16 h and inactivation of the enzyme by heating at 70 °C for 30 min. The samples were then separated on 1.2% agarose gel by electrophoresis and blotted onto a HybondTM-N+ membrane (GE Healthcare, Amersham, Buckinghamshire, UK). Biotin-labeled probes were obtained using North2South Biotin Random Prime DNA Labeling kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol, and used for hybridization. Hybridization was performed with a 30-min pre-hybridization at 55 °C for 30 min in the North2South Chemiluminescent Detection kit hybridization buffer (Thermo Fisher Scientific, Waltham, MA, USA) and subsequent hybridization at 55 °C overnight in hybridization buffer containing 30 ng/mL of the labeled probe. Membranes were washed, blocked, and equilibrated. Probe-target hybrids were visualized using Streptavidin:HRP conjugates and Peroxide/Luminol working solution.

Total RNA from cultured cells and sucrose gradient fractions were isolated with TRIzol according to the manufacturer’s instructions. All RNA samples to be used in reverse transcriptase (RT) reactions were treated with turbo DNase (Thermo Fisher), followed by re-isolation with TRIzol. All samples were re-precipitated with 0.1 volume of 3 M sodium acetate and 3 volumes of ice cold 100% ethanol. For northern blotting, 5 μg of total RNA from each sample was analyzed through 1.2% agarose-formaldehyde gels and was transferred to HybondTM-N+ membrane (GE Healthcare) by neutral transfer. T4 Polynucleotide Kinase (NEB) 5′ radiolabelled oligonucleotides were used for detection of mitochondrial transcripts (MT-ATP8/6 5′-TGGGTGATGAGGAATAGTGTAAGGAG; MT-CO3 5′-ATAGGCATGTGATTGGTGGGTCAT; MT-ATP6—MT-CO3 border 5′-ATTGGTGGGTCATTATGTGTTGTC). Hybridization (25% formamide, 7% SDS, 1% BSA, 0.25 M sodium phosphate pH 7.2 and 1 mm EDTA pH 8.0, 0.25 M NaCl2) was performed overnight at 37°C. Membranes were washed (2× SSC/0.1% SDS) and were then dried for exposure on a phosphorimager screen (GE Healthcare) and were scanned with a Typhoon 9400 (GE Healthcare).