Plasmids containing calsyntenin cDNA fragments (ESTs obtained from Source BioScience) were linearized by digestion with the appropriate restriction enzymes to produce templates for the synthesis of antisense and sense probes. The ESTs used were: ChEST846m5 for Clstn1, ChEST1002c5 for Clstn2 and ChEST882h15 for Clstn3. For linearization, 10 μg plasmid DNA were incubated with 20 U of the restriction enzyme in the appropriate buffer for 2–4 h at 37°C. After phenol/chloroform extraction and acetate/ethanol precipitation, DIG-labeled sense and anti-sense probes were synthesized by in vitro transcription. Two microgram linearized plasmid DNA, 2 μl of 10× concentrated DIG RNA Labeling Mix (Roche), 2 μl 100 mM DTT (Promega), 4 μl 5× concentrated transcription buffer (Promega), 1 μl RNasin (40 U/μl; Promega), 2 μl of T3 or T7 RNA polymerase (Roche) and diethyl pyrocarbonate (DEPC)-treated H2O were mixed to a final volume of 20 μl and incubated at 37°C for 2 h. The DIG-labeled RNA probes were extracted by lithium chloride precipitation and dissolved in 100 μl DEPC-treated H2O.
T3 or t7 rna polymerase
Manufactured by Roche
Sourced in Germany, United States
The T3 or T7 RNA polymerase is a bacterial enzyme used in molecular biology to transcribe RNA from DNA templates. It recognizes specific promoter sequences on the DNA and initiates the synthesis of RNA molecules. The enzyme is capable of producing large quantities of RNA transcripts with high fidelity and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Dig rna labeling mix, by Roche (3 mentions)
Rnasin, by Promega (1 mentions)
Dig rna labeling mixture, by Roche (1 mentions)
Dfc310 fx camera, by Leica (1 mentions)
Kod plus neo, by Toyobo (1 mentions)
Rna labeling mix, by Roche (1 mentions)
Qiafilter plasmid midi kit, by Qiagen (1 mentions)
Fluorescein rna labeling mix, by Roche (1 mentions)
Recombinant dnase 1, by Takara Bio (1 mentions)
Dig labeling mix, by Roche (1 mentions)
Pcr script amp cloning kit, by Agilent Technologies (1 mentions)
Purelink miniprep kit, by Thermo Fisher Scientific (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
8 protocols using t3 or t7 rna polymerase
1
Labeling Calsyntenin Transcripts for In-Situ Hybridization
2
Synthesis of DIG-labeled RNA Probes
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DIG-labeled cRNA probes used for Southern-blot or Northern-blot hybridization were transcribed from plasmid constructs including a full length SlartRDR6 sequence or a partial sequence of CaMV-35S promoter or a dimer of minus strand PSTVd-Dahlia (S1 and S2 Figs) [50 ]. The plasmid construct was digested with restriction enzymes and treated with phenol:chloroform (1:1, vol/vol). The ethanol precipitate was air-dried, suspended in ultrapure water, and used for in vitro transcription with T3 or T7 RNA polymerase and DIG RNA labeling mixture (Roche Diagnostics). The transcripts were collected by ethanol precipitation using LiCl, air-dried, and suspended in ultrapure water. Concentration and size of the probe was confirmed by agarose gel electrophoresis.
3
In Situ Hybridization of E18.5 Kidneys
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E18.5 kidneys were fixed in 4% PFA, dehydrated and stored in methanol at −20 °C. In situ hybridization using a digoxigenin-labeled riboprobe was performed as described97 (link). Probes were generated using the DIG RNA Labeling Mix (Roche, 11175025910) and T3 or T7 RNA polymerase (both Roche, 11031163001 or 10881767001). An alkaline phosphatase-conjugated antibody was used to detect the DIG-labeled probes (Roche, 11093274910).
4
Antisense RNA Probes for In Situ Hybridization
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To obtain templates of antisense DIG-labeled RNA probes, coding sequences of interests were amplified using KOD Plus Neo (TOYOBO) from cDNA and then cloned into SmaI-digested pBlueScript II SK(−) plasmid, followed by sequence validation (FASMAC). Antisense DIG-labeled RNA probes were synthesized using T3 or T7 RNA polymerase (Roche) and DIG RNA Labeling mix (Roche). At 7 dpf, larvae ware fixed in 4% PFA in PBS at 4°C overnight. Larvae were washed with PBS five times and treated with 3% hydrogen peroxide in 0.5% sodium hydride at room temperature to bleach pigments. After removing pigments, larvae were washed with PBS, and then transferred into methanol and stored at −30°C until staining. Whole-mount in situ hybridization was performed as described (Takada et al., 2017 (link)) with only a slight modification in that we performed proteinase K treatment by 10 µg/ml concentration at 25°C for 45 min. Images were taken with a Leica DFC310 FX camera.
5
In situ Hybridisation Localization of HvSUT1 and HvSUT2
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For in situ hybridisation, grains were fixed in 50% (v/v) ethanol, 5% (v/v) acetic acid, and 3.7% (w/v) formaldehyde overnight at 40 °C, dehydrated, and embedded in paraffin. Cross-sections (12μm) were de-waxed, rehydrated, and treated with 2μg ml−1 proteinase K for 30min at 37 °C. Tissue sections were dehydrated and dried before applying the hybridisation solution. Hybridisations were performed with 1 ng μl−1 digoxigenin-labelled sense and antisense RNA probes for HvSUT1 and HvSUT2-cDNA synthesised using T3 or T7 RNA polymerase (Roche, Germany). Hybridisation signals were detected by alkaline phosphatase-conjugated anti-digoxigenin antibody and visualised with 4-nitroblue tetrazolium-chloride and 5-bromo-4-chloro-3-indolyl phosphate (www.roche.de ).
6
Whole-Mount In Situ Hybridization
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For whole-mount in situ hybridization, the cDNA for each probe was cloned into pTA2 (Toyobo, Osaka, Japan) using the following forward and reverse primers: adam12, 5′-CTCCACTCATCACTCACCCG-3′ and 5′-CGATCTCCGCTAGCCTTTGT-3′; gata1a, 5′-ACACTTGCAGATGGAGAACTCCTC-3′ and 5′-GTGCCATGTGAGACATGTCAGG-3′; and hbae1 (hemoglobin embryonic subunit α), 5′-CCTTCCACAATGAGTCTCTCTGCC-3′ and 5′-GTACTTCTCAGACATGGCCAGAGC-3′. Antisense RNA probes were synthesized using T3 or T7 RNA polymerase (Roche Diagnostics, Indianapolis, IN, USA) and RNA labeling mix (Roche). In situ hybridization was performed according to a standard protocol in our laboratory.
7
Synthesis and characterization of zebrafish riboprobes
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Each zebrafish RT-PCR product was ligated into pBluescript II SK(−) vector. Sequences of primers used for RT-PCR are listed in Table 2 . Degenerate primers were designed to amplify several paralogs. After cloning and sequencing, the plasmids were extracted with the QIAfilter Plasmid Midi Kit (QIAGEN) and then linearized with the appropriate restriction enzyme. Digoxigenin (DIG)-labeled or fluorescein-labeled riboprobes were synthesized with T7 or T3 RNA polymerase (Roche) from the linearized plasmids with DIG or fluorescein RNA labeling mix (Roche), respectively. The riboprobes were treated with recombinant DNase I (TaKaRa) to exclude template plasmids.
8
Quantification of HOTAIR lncRNA Expression
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PCR on cDNA generated from total RNA of knee SF was used to produce an amplicon of 267 base pairs of HOTAIR (see Supplementary Table S4 ). Amplicons were cloned into pPCR-Script Amp SK (+) plasmids using the PCR-Script Amp cloning kit (Agilent Technologies). Plasmids were amplified and purified with the PureLink MiniPrep kit (Thermo Fisher Scientific). Plasmids were linearized using restriction enzymes (EcoRI or NotI; both New England Biolabs) and purified with the QIAquick PCR Purification Kit (Qiagen). DIG-labeled HOTAIR probes were prepared by in vitro transcription with RNA polymerases and plasmid vectors containing target transcript sequences. Linearized plasmid DNA (10 µl) was used as a template, and RNA probes were synthesized with T7 or T3 RNA polymerase (Roche) and DIG Labeling Mix (Roche) for 100 min at 37 °C.
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