Total RNA from minigenome assay cells was extracted with TRIzol (Thermo Fisher Scientific). Five microgram of total RNA was digested with RQ1 DNase (Promega) for 1 h at 37°C. The RNA samples were electrophoresed for 1 h at 100 V in a 1× MOPS and 6% formaldehyde agarose gel and transferred to Hybond-N+ membrane (GE Healthcare Life Sciences). Probes specific for genomic and RI RNAs (Table 1 ) were labeled with 32P ATP using T4 Polynucleotide Kinase (Promega). Membranes were prehybridized for 1 h in PerfectHyb Plus Buffer (Sigma-Aldrich), and probe was added and hybridized overnight at 45°C for genomic and 55°C for RI, respectively. Membranes were exposed to phosphor screen overnight, imaged on GE Typhoon FLA 9500 variable mode imager and quantified with ImageQuant software (GE Healthcare). Signal was normalized to VP35(-) sample for the genomic probe and normalized to wt sample for the RI probe.
Perfecthyb plus buffer
Manufactured by Merck Group
Sourced in United States
PerfectHyb Plus buffer is a hybridization buffer designed for use in molecular biology applications. It is formulated to promote efficient and specific hybridization of nucleic acid probes to target sequences. The buffer contains detergents, salts, and other components that help to minimize non-specific interactions and improve signal-to-noise ratios during hybridization experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Tri reagent, by Merck Group (2 mentions)
Hybond nx, by Cytiva (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Amersham hybond n membrane, by GE Healthcare (2 mentions)
Hybond n, by Cytiva (2 mentions)
Imagequant software, by GE Healthcare (1 mentions)
T4 polynucleotide kinase, by Promega (1 mentions)
Rq1 dnase, by Promega (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Typhoon fla 9000 scanner, by GE Healthcare (1 mentions)
Phosphorimager screen, by Fujifilm (1 mentions)
α 32p datp, by Hartmann Analytic (1 mentions)
Decaprime 2 kit, by Thermo Fisher Scientific (1 mentions)
Multi gauge v3, by Fujifilm (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
Typhoon multimode imager, by GE Healthcare (1 mentions)
Hybond n, by GE Healthcare (1 mentions)
Prime a gene labeling system, by Promega (1 mentions)
Precellys 24 homogenizer, by Bertin Technologies (1 mentions)
13 protocols using perfecthyb plus buffer
1
Northern Blot Analysis of Minigenome RNA
2
Quantification of Mitochondrial Transcripts
3
Detection of BBSV and sat-RNA via Northern Blot
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Total RNA was extracted using TRIzol® Reagent (Invitrogen, USA). For detection of viral genomic RNA and mRNA of BBSV and its sat-RNA, 2 μg total RNA extracted from mock or virus-infected plants was used for hybridization using indicated gene-specific 32P-radiolabled cDNA probes corresponding to the 3′UTR fragment of BBSV or the full-length sat-RNA, respectively as described (Xu et al., 2012 (link)). For small RNA gel blots, 10 μg total RNA were separated on 17% denaturing polyacrylamide gel (PAGE) and transferred to nylon membranes (GE Healthcare, UK). DNA oligonucleotides corresponding to the sequences of BBSV (nt 155–176, nt 769–788, nt 823–842, nt 1259–1278, nt 1762–1781, nt 2020–2039, nt 2266–2287, and nt 3115–3134) or sat-RNA (nt 132–152, nt 395–416 and nt 506–527) were synthesized respectively. The mixtures of antisense oligonucleotides corresponding to BBSV and sat-RNA were labeled with [γ-32P] ATP as probes used for hybridization at 40°C for 16–20 h in PerfectHyb plus buffer (Sigma-Aldrich). The membranes were washed in 2 × SSC (0.3 M NaCl and 0.03 M sodium citrate) containing 0.2% SDS for 30 min and then twice with 1 × SSC containing 0.1% SDS for 20 min both at 50°C.
4
Small RNA Isolation and Detection
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Total RNA was extracted from Arabidopsis inflorescences using TRIzol reagent (Invitrogen) and size-fractionated as described in (53 (link)). Then, 9 μg low molecular weight RNA was resuspended in 8 μl RNA Loading Buffer (95% formamide, 0.025% bromophenol blue, 0.025% xylene cyanol FF, 5 mM EDTA, 0.025% SDS, pH 8.5). Samples were heated to 95°C for 3 min and separated on an 16% polyacrylamide gel. RNA loading was documented using Ethidium bromide gel staining followed by UV transillumination. Size-separated RNAs were transferred to a nylon membrane (Hybond-N+, GE Healthcare) by electroblotting and UV cross-linked (140 mJ/cm2). Different 32P 5′-end-labeled DNA oligonucleotides were used for successive hybridizations in PerfectHyb Plus Buffer (Sigma) overnight at 35–40°C, depending on the probe. The membrane was washed three times for 20 min in Wash Buffer (0.3 M NaCl, 30 mM sodium acetate, 0.5% SDS, pH 7.0), exposed to a phosphor-imager screen for 3 days, then the screen was scanned using a Typhoon Multimode-imager (GE Healthcare). Each probe was stripped with boiling 0.1% SDS (two times, 20 min) prior to the next hybridization (see Supplementary Table S1 for probe sequences).
5
Small RNA Northern Blot Analysis
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Total RNA was extracted using the TRIzol® reagent (Invitrogen™) according to the manufacturer’s instructions. For small RNA Northern blot analysis, total RNAs were fractionated in a 17.5% polyacrylamide gel containing 8 M urea. As probes we used γ32P-ATP end-labelled oligonucleotides complementary to the small RNA sequence under study. Probes used for Northern blot analysis are indicated in Additional file 1 : Table S3. Blots were pre-hybridized and hybridized in Perfect-Hyb Plus buffer (Sigma). Hybridization signals were detected using a STORM Phosphorimager (GE Helthcare).
6
RNA Extraction and Northern Blot Analysis
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Equal amounts of deep-frozen leaf material were ground in a Precellys 24 homogenizer (Bertin Technologies). Total RNA was extracted using TRI-Reagent (Sigma-Aldrich) according to the manufacturer’s instructions and solubilized in sterile water. For northern blot analysis, 5 μg of total RNA was separated by denaturing gel electrophoresis, transferred to a nylon membrane (Hybond-N; Amersham Biosciences AB), cross-linked with UV light, prehybridized in PerfectHyb Plus buffer (Sigma-Aldrich) at 60 °C for 30 min and incubated overnight with the radioactive probe. The radioactive probe was generated using the Prime-a-Gene labeling system (Promega) with a PCR product corresponding to the 5′ 200 bases of GFP as template. After hybridization and washing, membranes were exposed to X-ray film for autoradiography.
7
RNA Isolation and Blot Analysis
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High molecular weight (HMW) and low molecular weight (LMW) total RNA was isolated for gel blot analysis as described previously (Mlotshwa et al., 2005 ). For mRNA gel blot analysis, 5 µg of HMW RNA was run on formaldehyde denaturing agarose gels; and for siRNA gel blot analysis, 10 µg of LMW RNA was run on 20% polyacrylamide/7 M urea denaturing gels. The resolved RNA (HMW or LMW) was blotted onto Amersham hybond‐N+ membranes (GE Healthcare) and hybridized with RNA probes. PDS probes were derived from cDNA fragments corresponding to the maize PDS mRNA sequence (GenBank Accession # NM_001352010.1; nt 1493–1612 and 1613–1732 for siRNAs and mRNA probes, respectively). LSP probes were derived from cDNA fragments corresponding to the maize LSP mRNA sequence (NCBI sequence NM_001175829; nt 1217–1426 and 1427–1651 for siRNAs and mRNA probes, respectively). The respective cDNA fragments were PCR‐appended with a T7 promoter at the 3′‐ or 5′‐end to make RNA probes to detect mRNAs or antisense siRNAs, respectively. The [α‐32P]UTP‐labeled RNA probes were synthesized using the MAXIscript T7 in vitro transcription kit (Invitrogen) and hybridized to mRNA blots at 68°C, or to siRNA blots at 42°C in PerfectHyb Plus buffer (Sigma). The siRNA sizes were based on Decade molecular weight RNA markers (Invitrogen) labeled with gamma‐32P‐ATP.
8
Murine Genomic DNA Isolation and Southern Blot Analysis
9
Profiling Rice miRNA Expression
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Total RNA was extracted from plant tissues by using TRizol reagent (Invitrogen). For northern blot analysis of rice miRNAs, RNAs were fractionated in a 17.5% denaturing polyacrylamide gel containing 8 M urea, transferred to nylon membranes and probed with a γ32P-ATP end-labeled miR7695.3-3p oligonucleotide (Additional file 2 : Table S1). Blots were pre-hybridized and hybridized in Perfect-Hyb Plus buffer (Sigma) at 42 °C. Hybridization signals were detected by using STORM Phosphorimager (GE Healthcare).
For quantitative RT-PCR (RT-qPCR), the first complementary DNA was synthesized from DNase-treated total RNA (1 μg) with High Capacity cDNA Reverse Transcription (Life technology, Applied Biosystems). Amplification involved 2 μl cDNA (5 ng/μl) in optical 96-well plates (Roche Light Cycler 480; Roche Diagnostics, Mannheim, Germany) with SYBR Green I dye and gene-specific primers (Additional file2 : Table S1). The Ubiquitin1 gene (Os06g0681400) was used to normalize transcript levels.
For quantitative RT-PCR (RT-qPCR), the first complementary DNA was synthesized from DNase-treated total RNA (1 μg) with High Capacity cDNA Reverse Transcription (Life technology, Applied Biosystems). Amplification involved 2 μl cDNA (5 ng/μl) in optical 96-well plates (Roche Light Cycler 480; Roche Diagnostics, Mannheim, Germany) with SYBR Green I dye and gene-specific primers (Additional file
10
Northern Blot Analysis of miRNAs
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Twelve μg of total RNA from apices of D7 seedlings were separated on 17.5% PAGE-urea gels, blotted, and cross-linked to Hybond NX (Amersham ref. RPN203T) nylon membrane, as previously described (Incarbone et al. 2018 (link)). Probe hybridization was performed in PerfectHyb Plus buffer (Sigma ref. H7033) overnight at 42 °C, followed by 3 15-min washes in 2×SSC 2% SDS at 48 °C. miRNA160 and U6 probes were obtained by labeling DNA oligonucleotides via a PNK reaction with γ32ATP. To detect transposon-derived siRNA, PCR products were labeled with α32CTP through Klenow reaction. All primers and oligos used for the synthesis of probes are listed in Supplemental Data Set 5 .
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