Approximately 15 µg of total RNA (prepared as described above) was individually separated in a 15% urea polyacrylamide gel, electrophoretically transferred to Hybond-NX membrane (GE Healthcare, Buckinghamshire, UK) using a semi-dry transfer apparatus (Amersham Biosciences, Piscataway, NJ), and was chemically cross-linked via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) [64] (link). For labeling reaction of probes, 1 µl of 10 µM probes, 2.5 µl of 10 x T4 PNK buffer (New England Biolabs), 3 µl of [γ-32P] ATP (∼10 µCi/µl), 17.5 µl of ddH2O and 1 µl of T4 Poly Nucleotide Kinase (New England Biolabs) were added (a total volume of 25 µl reaction) and kept in a water bath for 1 hour at 37°C. Probe sequences used for Northern blot analysis were shown in Table S7 . Blots were pre-hybridized and hybridized at 42°C overnight using hybridization buffer (Sigma, USA). Post-hybridization washes were performed using 2 x SSC and 0.2% sodium dodecyl sulfate (SDS) at 42°C for 20 min for twice. Hybridization signals were detected by exposing blots to autoradiographic film.
Hybridization buffer
Manufactured by Merck Group
Sourced in United States
Hybridization buffer is a solution used in molecular biology and genetic analysis to facilitate the hybridization of nucleic acid molecules, such as DNA or RNA. It provides the necessary chemical environment for the specific binding of complementary nucleic acid sequences during various hybridization techniques.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Semi dry transfer apparatus, by Cytiva (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Hybond nx membrane, by GE Healthcare (1 mentions)
T4 pnk buffer, by New England Biolabs (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
Trans blot sd semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Uvc 500 crosslinker, by Hoefer (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Prime a gene labeling system, by Promega (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hybond n membrane, by Cytiva (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Nylon membrane, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Lysozyme solution, by Thermo Fisher Scientific (1 mentions)
8 protocols using hybridization buffer
1
Northern Blot Analysis of RNA
2
Northern blot analysis of miRNA
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Total RNA was isolated from the tissue samples of the control plants and 340 mM NaCl-treated (9 h) S. maritima using miRNeasy mini kit (Qiagen) according to the manufacturer’s instructions. For the Northern blot analysis, 10 μg of total RNA was resolved on a 15 % urea-PAGE gel. The electrophoresed RNA was transferred onto a nylon membrane using a Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The blot was air dried and UV cross-linked at 150 mJ using a UV cross-linker (Hoefer™ UVC 500 Crosslinker). Probes designed from DNA oligonucleotides complementary to the miRNA sequences (Additional file 2 ) were end-labeled with [γ-32P]dATP using T4 Polynucleotide Kinase (Fermentas) according to the manufacturer’s instructions. The membrane was pre-hybridized for 1 h with hybridization buffer (Sigma), and then the labeled probe was added and allowed to hybridize for 16 h at 37 °C. After hybridization, the membrane was washed with 2X SSC and 0.1 % SDS at 32 °C for 15 min and 1X SSC and 0.1 % SDS for 15 min at 37 °C. The membrane was air dried and then exposed to X-ray film. When required, the membrane was stripped, re-exposed to the X-ray film to ensure complete signal removal and reused for a second hybridization. A DNA oligonucleotide complementary to U6 snRNA was used as a probe to detect the U6 snRNA for use as an internal control.
3
Immunostaining and FISH for Intestinal Tissues
4
Plant RNA Extraction and Analysis
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Plant total RNA used for RNA and sRNA gel blots was extracted using Trizol Reagent (Invitrogen) according to the manufacturer's protocol. Isolated RNA was reverse transcribed using oligo (dT) primer HC51118TR and Moloney murine leukemia virus (M‐MLV) reverse transcriptase (Promega). Semiquantitative reverse transcriptase PCR (RT‐PCR) was performed as described previously (Wang Y. et al., 2013 ).
Total RNA was separated in a 1.5% denaturing gel and then blotted to Hybond‐N+ membrane (Amersham Pharmacia Biotech). Templates of probes were amplified by PCR with respective primers and labeled with [α‐32P] dCTP using the Prime‐a‐Gene Labeling System (Promega) in hybridization buffer (Sigma‐Aldrich) at 65°C for 16 h. The signals were detected by exposing the membrane to X‐ray film with the chemical fluorescent substrate. Signal intensity was quantified usingimagequant tl software (GE Healthcare, Little Chalfont, UK).
Total RNA was separated in a 1.5% denaturing gel and then blotted to Hybond‐N+ membrane (Amersham Pharmacia Biotech). Templates of probes were amplified by PCR with respective primers and labeled with [α‐32P] dCTP using the Prime‐a‐Gene Labeling System (Promega) in hybridization buffer (Sigma‐Aldrich) at 65°C for 16 h. The signals were detected by exposing the membrane to X‐ray film with the chemical fluorescent substrate. Signal intensity was quantified using
5
Fluorescence in situ hybridization protocol
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Fluorescence in situ hybridization was performed as described in the ref. 10 . Briefly, cells were fixed with 4% paraformaldehyde for 15 min then permeabilized with −20 °C methanol for 5 min. Cells were incubated overnight in 70% ethanol at 4 °C. The following day, cells were washed twice with 2× saline-sodium citrate (SSC, Ambion, Waltham, MA USA), blocked in hybridization buffer (Sigma, Saint Louis, MO, USA) for 30 min. Hybridization was performed using 2 ng/µl of biotinylated oligo-dT40× probe diluted in hybridization buffer at 37 °C. After extensive washes with 2 × SSC at 37 °C the probe was revealed using a fluorophore-conjugated streptavidin antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA), followed by immunostaining as described above.
6
miR-205 Expression Analysis in Bladder Cells
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Expression of miR-205 in bladder cell lines were examined by northern blot using a 32P-labeled oligo (5′-CAGACTCCGGTGGAATGAAGGA-3′). Briefly, total RNA was extracted from cells using Trizol (Life Technology), 30 μg of total RNA was heated at 70 °C for 5 min and electrophoresized in a 15% denaturing PAGE gel, then transferred to a nylon membrane (BioRad), prehybridized in hybridization buffer (Sigma-Aldrich, Natick, MA, USA) for 1 h at 37 °C, and then hybridized overnight with the 32P-labeled oligo at 40 °C. MiR-205-radiolabelled probes were removed from membranes by washing twice with and the hybrid signals are exposed to a high-sensitive X-ray film overnight. An antisense oligo of U6 (5′-AAAATATGGAACGCTTCACGA-3′) was used to detect U6 snRNA from each sample as a loading control.
7
Fluorescent Biofilm Staining and Imaging
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Coupon biofilms samples were washed with PBS (Thermo Fisher Scientific, MA, USA), fixed in 4% paraformaldehyde (Sigma Aldrich, UK) for 2 h at 4°C, washed again with PBS and 1 mL of dehydration buffer (50% ethanol in PBS), and placed at −20°C for 2 h. Subsequently, the samples were washed with PBS and incubated in 1 mL lysozyme solution (1 mg mL−1) (Thermo Fisher, USA) for 15 min at 37°C. After another wash with PBS, hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl pH 7.2, 0.01% vol/vol SDS, and 25% vol/vol formamide) (Sigma Aldrich, UK) and 250 ng of the appropriate DNA probe (see Table 4 ) were added. The biofilms were incubated in the dark covered in aluminum foil for 3 h at 46°C. After incubation, the biofilms were washed with a wash buffer (10 mM Tris-HCl pH 9.0 and 1 mM EDTA) (Sigma Aldrich, UK) and incubated in this buffer at 55°C for 10 min. This step was repeated three times. All biofilms were kept in the dark until imaged with the Zeiss LSM880 confocal laser scanning microscope (Zeiss, Germany). Excitation intensity and any post-processing using the Zen Black software (Zeiss, Germany) were recorded within the software for accurate depiction of signal received.
8
RNA Fluorescence In Situ Hybridization in U2OS Cells
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U2OS cells were treated as indicated in figure legends then fixed with 4% paraformaldehyde for 15 min, permeabilized with −20°C methanol for 10 min, then dehydrated in 70% ethanol at 4°C. Cells were stored at 4°C for at least overnight then wash several times with 2X saline sodium citrate (SSC; Ambion). Cells were equilibrated in hybridization buffer (Sigma) for 15 min at 65°C then incubated with oligo dT40X probe for 1 h at 42°C. Cells were washed twice with 2X SSC for 10 min at 42°C then several times at room temperature. Coverslips were then processed for immunofluorescences as indicated above (from block step on).
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