For RNA in situ hybridization, spikelet hulls were fixed in FAA solution for 24 h at 4 °C, then dehydrated using a graded ethanol series, followed by a xylene series, and embedded in Paraplast Plus (Sigma‐Aldrich, St Louis, USA) for sections. The sense and antisense RNA probes used in this test are listed in Table S3. Digoxigenin‐labelled RNA probes were prepared using a DIG RNA Labelling Kit (SP6/T7) (Roche, Mannheim, Germany) according to the manufacturer's instructions. A sense probe was used as a negative control.
Dig rna labelling kit sp6 t7
Manufactured by Roche
Sourced in Germany, United States
The DIG RNA Labelling Kit (SP6/T7) is a tool used in molecular biology research to generate digoxigenin-labeled RNA probes for nucleic acid detection and analysis. The kit provides the necessary reagents and enzymes to transcribe and label RNA from DNA templates containing SP6 or T7 promoters.
Automatically generated - may contain errors
Lab products found in correlation
Paraplast plus, by Merck Group (2 mentions)
Criterion tbe urea polyacrylamide gel, by Bio-Rad (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Rm2135 microtome, by Leica (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Genelute plasmid miniprep kit, by Merck Group (1 mentions)
Ab 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator reaction chemistry, by Thermo Fisher Scientific (1 mentions)
M165 fc stereomicroscope, by Leica (1 mentions)
Bm purple, by Roche (1 mentions)
Nbt bcip reagents, by Roche (1 mentions)
Dig northern starter kit, by Roche (1 mentions)
Perfection v700, by Epson (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Hybond nylon membrane, by Cytiva (1 mentions)
Hybond, by Cytiva (1 mentions)
Dm6b microscope, by Leica (1 mentions)
Pgem t easy ta cloning vector, by Promega (1 mentions)
15 protocols using dig rna labelling kit sp6 t7
1
RNA in situ Hybridization in Spikelet Hulls
2
In Situ Hybridization of SRL2 Gene
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Leaves were collected from fourth leaf stage of the wild type and fixed in FAA overnight at 4 °C. Samples were then dehydrated through a butanol series and embedded in Paraplast Plus (Sigma–Aldrich, http://www.sigmaaldrich.com/ ). Sections, 8 μm thick, were obtained using a Leica RM2135 microtome (Leica Biosystems, http://www.leica.com ). To prepare the SRL2 probe, a 492-bp fragment of SRL2 cDNA was amplified using primers SRL2 T7 and SRL2 sp6 (SupplementaryTable S2 ). The probe was synthesized using a DIG RNA labelling kit (SP6/T7; Roche Diagnostics Ltd, http://www.roche.com ) in accordance with the manufacturer’s recommendations. Pretreatment of sections, hybridization, and immunological detection were performed as described previously (Xiao et al., 2009 (link)).
3
In Situ Hybridization with RNA Probes
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cDNA was produced with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) based on the manufacturer’s instructions and used to amplify ~ 500 bp long templates. Probes were synthesized with DIG RNA Labelling Kit (SP6/T7) (Roche) according to the manufacturer’s instructions. In situ hybridization was performed as described by White-Cooper with the following differences: hybridization buffer contains the additional 100 μg ml− 1 tRNA (Sigma) [73 ]. Images were taken by using Olympus BX51 microscope.
4
Cloning and in situ Hybridization of fhl2a and fhl2b in Astatotilapia burtoni
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A. burtoni fhl2a and fhl2b coding fragments were amplified by PCR (for primer information, see Supplementary Table 3 ) using Phusion Master Mix with High Fidelity buffer (New England BioLabs, USA) following the manufacturer’s guidelines. These fragments were cloned into pCR4-TOPO TA vector using the TOPO TA cloning kit (Invitrogen). Plasmid extractions were done with GenElute Plasmid Miniprep Kit (Sigma-Aldrich). RNA probes were synthetized with the DIG RNA labelling kit (SP6/T7) (Roche). The insertion and direction of the fragments was confirmed by Sanger sequencing using M13 primers (available with the cloning kit) and BigDye terminator reaction chemistry (Applied Biosystems) on an AB3130xl Genetic Analyzer (Applied Biosystems). In situ hybridization was performed in 12 fins from A. burtoni males, six for fhl2a and six for fhl2b. The protocol was executed as described in ref. 16 (link), except for an intermediate proteinase K treatment (20 min at a final concentration of 15 μg ml−1) and for the hybridization temperature (65 °C).
5
Spatiotemporal Gene Expression Profiling
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The following riboprobes were used: fgf8 (fibroblast growth factor 8), gad1b (glutamate decarboxylase GAD67), neuroD (neurogenic differentiation), notch1a (notch homologue 1a), pax2a (paired box gene 2a), pax6a (paired box gene 2a), shh a (sonic hedgehog a) and wnt1 (wingless-type MMTV integration site family, member 1a). The probes were transcribed from their templates using the DIG RNA Labelling Kit (SP6/T7) (Roche Applied Science) following the manufacturer's instructions. In situ hybridisations were performed on wholemounts as previously described [35] (link), and probes were detected using BM purple or NBT/BCIP reagents (Roche Applied Science). Control experiments were always performed in parallel. Images were taken with a Leica M165FC Stereomicroscope. 25 embryos were scored for each treatment and for each time point, and each experiment was repeated three times.
6
RNA Detection through Northern Blot
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Total RNA was isolated from the two RNAi lines and control plants using the TRIzol method. Samples of total RNA (80 μg) were resolved by 15% polyacrylamide/0.5 X TBE urea gels and then blotted onto a BrightStar™-Plus positively charged nylon membrane (15 cm × 15 cm). For probe preparation, primers for the complete sense strand were used to isolate the gene sense strand suitable for siRNA. To generate specific DNA probes (590 bp), digoxigenin labelling was performed using a DIG-RNA labelling kit (SP6/T7) (Roche). Hybridization was performed using a DIG Northern starter kit (Roche) according to the manufacturer’s instructions, which involved blocking the membrane and immunological detection with an anti-DIG-AP conjugate. Signals were detected by the chemiluminescence method using CDP Ready-To-Use solution (Applera Corporation) as the substrate.
7
Gene Expression Profiling via WISH
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WISH was performed according to standard protocols [29 ]. The following probes were used: 881 bp p63 probe (nucleotides 875 to 1,755, NCBI Reference Sequence: NM_001127261.1), 1,034 bp Jag2 probe (nucleotides 592 to 1,625, NM_010588.2), and 473 bp Msx2 probe (nucleotides 94 to 566, NM_013601.2). The nucleotide sequence of the probe for Fgf8 [30 (link)] was generously provided by Prof. Hiroki Kurihara (The University of Tokyo, Tokyo, Japan). Digoxigenin (DIG)-labelled riboprobes for these genes were generated by a DIG RNA labelling kit (SP6/T7) (Roche, Basel, Switzerland).
8
Synthesis of Digoxigenin-Labeled RNA Probes
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Digoxigenin (DIG) labelled, single-stranded RNA probes were synthesised with T7 or SP6 polymerase using the Roche DIG RNA Labelling Kit (SP6/T7) (ROCHE 11175025910, Mannheim, Germany), following the manufacturer’s instructions. The reaction was incubated at 37 °C for 2 h, then 2 μL of RNase-free DNase I was added and the reaction was incubated for an additional 20 min. Last, the reaction mixture was transferred to a 1.5 mL RNase-free tube, and 4 μL of 0.2 M EDTA was added to stop the reaction. Formulations of the reagents used are listed in Supplementary Table S1 .
9
Detecting Virus-Derived Small RNAs
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Virus derived small RNAs were detected using Northern blotting. Forty micrograms total RNA were fractionated on 15% Criterion TBE-Urea polyacrylamide gel (Bio-Rad, Hercules, CA, USA) at 100V for 2 h. RNA was electrotransferred onto positively charged Hybond nylon membrane (Amersham, UK) using trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA) at 25V for 30 min and immobilized by crosslinking to the membrane twice at 120,000 microjoules/cm2 using a Stratalinker UV crosslinker 1800 (Stratagene, La Jolla, CA, USA). RNAs probes for virus derived siRNAs detection were PCR amplified from AC2/AC3 region of each virus genome using primers listed on Table 1. In vitro transcription was performed using DIG RNA labelling kit SP6/T7 (Roche, Indianapolis, IN, USA) following manufacturer's instructions. The ensuing steps were performed as described by Kuria et al. (2017) . Blots were exposed to Amersham high-performance chemiluminescence film (GE Healthcare, Pittsburgh, PA, USA) for 15 min and processed on an automated developer (Konica Minolta-SRX-101A). The autoradiographs were scanned on Epson Perfection V700 photo scanned (Epson, CA, USA) and the signal quantified on image J.
10
Generation and Validation of Gene-Specific Probes for WISH
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The gene-specific probes used in WISH were cloned by PCR into a pGEM-T Easy vector (Promega, Madison, WI, USA) using the primers listed in Table S2 . Antisense probes were created in vitro using a DIG RNA Labelling kit (SP6/T7; Roche Applied Science, Penzberg, Germany). WISH was performed per the procedure described in our previous study [40 (link)].
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