ISH probes were designed to span 500–1000 base pairs and were targeted to CDS and/or UTR regions of each mRNA (see Table S2 ). Probes were designed to minimize cross-hybridization with off-target mRNAs, which was assessed using BLAST. For the detection of specific ORs, probes targeting multiple gene regions were typically generated and tested. Probe sequences were amplified by PCR using specific primers (Table S2 ), inserted into the pCRII-TOPO vector (ThermoFisher), and confirmed by restriction analysis and sequencing. DIG- and FITC-labeled antisense RNA probes were generated from 1 μg of linearized plasmid template using T7 or Sp6 RNA polymerases (NEB) and DIG-11-UTP (Roche) or FITC-11-UTP (Roche), treated with DNaseI (Promega), ethanol precipitated, and dissolved in a 30 μL volume of water.
T7 or sp6 rna polymerase
Manufactured by New England Biolabs
Sourced in United States
T7 or SP6 RNA Polymerase is an enzyme that catalyzes the synthesis of RNA from a DNA template. It is a key tool for in vitro transcription, allowing the production of RNA molecules from a DNA sequence containing the appropriate promoter.
Automatically generated - may contain errors
Lab products found in correlation
Pcrii topo vector, by Thermo Fisher Scientific (2 mentions)
Dig 11 utp, by Roche (2 mentions)
Fitc 11 utp, by Roche (2 mentions)
Dnase 1, by Promega (2 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Digoxigenin 11 utp, by Roche (2 mentions)
Dig rna labeling kit, by Roche (2 mentions)
Rnase a, by New England Biolabs (1 mentions)
Opti mem 1 medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Tripletof 5600, by AB Sciex (1 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
Nanolc ultra 1d plus, by AB Sciex (1 mentions)
T3 rna polymerase, by Roche (1 mentions)
Typhoon 9400 imager, by GE Healthcare (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Flexi rabbit reticulocyte lysate, by Promega (1 mentions)
Chromatide alexa fluor 488 5 utp, by Thermo Fisher Scientific (1 mentions)
Digoxigenin 11 utp, by Merck Group (1 mentions)
12 protocols using t7 or sp6 rna polymerase
1
Designing and Validating ISH Probes
2
Plasmid Topoisomer Relaxation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmid topoisomers were analyzed for the relaxation of superhelical turns using agarose-chloroquine gel electrophoresis in 1.75% agarose gels run in 40 mM Tris, 50 mM potassium acetate, 1mM EDTA, pH 8.3, containing sufficient chloroquine to resolve individual topoisomers. Transcription reactions were performed basically as described [20 (link)] using T7 or SP6 RNA polymerases (New England Biolabs, Ipswich, MA, USA) in RNAPol buffer supplemented with 0.5 mM ATP, UTP, GTP, and CTP, 40 mM KCl and 20 µg/mL RNaseA, for 1–2 hr at 37 or 40 °C. Samples were precipitated with two volumes of ethanol and redissolved and digested with RNaseA H (New England Biolabs, Ipswich, MA, USA) as recommended.
3
In Vitro Transcription of Capped RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids digested with XhoI were used as templates for in vitro RNA transcription with T7 or Sp6 RNA polymerases (New England Biolabs). With the exception of IGR CrPV luc, all in vitro produced RNAs were capped by adding the m7G(5′)ppp(5′)G cap analog to the transcription mixture. The transcription mixtures were treated with DNAse I and used directly for transfection. BHK or C6/36 subconfluent cells grown in 24-well plates were transfected (per well) with a mixture of 1 µg RNA and 2 µL Lipofectamine 2000 in 200 µL Opti-MEM I medium (both from Invitrogen). Experiments to analyze the translation of sgRNAs were performed in Opti-MEM I medium. For those experiments requiring a longer duration, the transfection medium was changed after 2 h to DMEM with 10% FCS for BHK cells or M3 with 10% FCS for C6/36 cells.
4
Designing and Validating ISH Probes
5
In situ probe generation from hippocampus cDNA
6
In situ probe generation from hippocampus cDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primers were used to create in situ probes using whole hippocampus cDNA as a template: Pafah1b1: forward 5’-AAAATGGTGCTGTCCCAGAG-3’ & reverse 5’-ACACGGAGGGTCTTGTCATC-3’, Med8: forward 5’-GACCTGGCCCTCTGTCCT-3’ & reverse 5’-CTTGCCGAAAGCCTGTGT-3’, Hist1h4j: forward 5’-TCATGTCTGGCAGAGGTAAGG-3’ & 5’-TTGCATCCGAGACAGCATAG-3’. Primers were designed using Primer 3 58 . PCR products were cloned into pCRII-TOPO using the TOPO TA Cloning Kit (Invitrogen). Insert sequences were verified with Sanger sequencing. Based on insert orientation, NotI or SpeI restriction endonucleases (NEB) were used to linearize the vector prior to in vitro transcription. Probes were synthesized using digoxigenin-11-UTP (Roche) and T7 or SP6 RNA Polymerase (NEB) according to the manufacturer’s instructions. Template DNA was removed with TURBO DNase (Applied Biosystems) according to the manufacturer’s instructions.
7
lnc-CTSLP4 Interactome Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro transcription of lnc-CTSLP4 full-length sense, antisense, and serial truncated sequences were performed using T7 or SP6 RNA Polymerase (New England Biolabs, Ipswich, Suffolk, England) according to the manufacturer’s instructions (T7 RNA polymerase was used to perform in vitro transcription of lnc-CTSLP4 full-length sense and SP6 RNA polymerase was used for lnc-CTSLP4 full-length antisense). RNA pulldown assays were performed with the Pierce Magnetic RNA-Protein Pull-Down Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). lncRNA-interacting proteins were obtained and subjected to MS analysis. LC-MS/MS detection was carried out on a hybrid quadrupole-TOF mass spectrometer (TripleTOF 5600+, SCIEX) equipped with a nanoLC system (nanoLC-Ultra 1D Plus, Eksigent).
8
Spatiotemporal Analysis of miRNA and mRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Timed pregnant mouse embryonic brains were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). The fixed tissues were cryoprotected with 25% sucrose in PBS and equilibrated in the O.C.T. Compound (Sakura) for 15–30 min before freezing. Sixteen-micron cryosections were generated and stored at −20 °C. Probes of miR-214, miR-214* and miR-124 labelled with digoxigenin (DIG) for in situ hybridization were purchased from Exqion. For the synthesis of probes of the protein coding genes, the unique sequences of the genes were cloned into the pGEM-T vector. The digoxigenin (DIG)-labelled antisense and sense cRNA probes were synthesized using T7 or SP6 RNA polymerase (New England Biolabs) or T3 RNA polymerase (Roche Diagnostics) in vitro with the corresponding plasmids using the DIG RNA Labeling Kit (Roche Diagnostics) according to the manufacturer’s instructions. The QK probes were designed from the unique coding sequences of the three mRNA isoforms respectively. The ISH procedure was performed as described21 (link).
9
Synthesis of Digoxigenin-Labeled ash2l Probe
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedures for ISH and ash2l probe sequence were the same as previously described (Gray et al., 2004; Zhou et al., 2017) . The digoxigenin labeled ash2l probe were synthesized using T7 or SP6 RNA polymerase (New England Biolabs) in vitro with pGEM-T plasmids using the DIG RNA Labeling Kit (Roche Diagnostics) according to the manufacturer's instructions.
10
In Vitro Synthesis and Translation of Capped, Labeled RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synthesis of capped RNA in vitro was accomplished with SP6 or T7 RNA polymerase (New England Biolabs, Ipswich, MA, USA), and m7G(5′)ppp(5′)G RNA cap analog (New England Biolabs) as described previously [9 (link)]. Synthetic RNA incorporating fluorescent or DIG labels were made using ribonucleotides mixed with ChromaTide® Alexa Fluor® 488-5-UTP- (Thermo Fisher Scientific) or Digoxigenin-11-UTP- (Sigma-Aldrich, Burlington, MA, USA) during in vitro transcription as per manufacturers’ protocol. The free labels were removed by Sephadex G-50 quick spin columns (Sigma-Aldrich).
In vitro translation was carried out using the Flexi® Rabbit reticulocyte lysate and the nonradioactive FluoroTect™ GreenLys in vitro translation labeling as per the manufacturer’s protocol (Promega, Madison, WI, USA). In brief, approximately 100–200 ng RNA transcripts were used in a 15 μL-reaction with indicated recombinant RuV proteins. The results were resolved on 10 or 12% SDS-polyacrylamide gels and visualized using a Typhoon 9400 Imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Data analysis was accomplished with ImageQuant software (GE Healthcare Life Sciences).
In vitro translation was carried out using the Flexi® Rabbit reticulocyte lysate and the nonradioactive FluoroTect™ GreenLys in vitro translation labeling as per the manufacturer’s protocol (Promega, Madison, WI, USA). In brief, approximately 100–200 ng RNA transcripts were used in a 15 μL-reaction with indicated recombinant RuV proteins. The results were resolved on 10 or 12% SDS-polyacrylamide gels and visualized using a Typhoon 9400 Imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Data analysis was accomplished with ImageQuant software (GE Healthcare Life Sciences).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!