T7 sp6 rna polymerase

The entire coding regions of miple1 and miple2 were cloned into the dual PCR II promoter TOPO vector (Invitrogen) and used as template to generate DIG-labeled anti-sense and sense probes using SP6/T7 RNA polymerase (Roche) and NTP/DIG-UTP mix (Roche). In situ hybridisation of larval tissues was performed according to [56] (link), while for adult brains a protocol including Proteinase K treatment was employed [57] (link). Samples were mounted on polylysine coated slides and visualised using DIC with Zeiss Axio Imager.Z2 microscope and Axio vision Release 4.8 software.

WISH was performed as previously described (Thisse and Thisse, 2008 (link)). Digoxigenin-labeled riboprobes for the four stag genes were synthesized from PCR clones inserted into pGEM®-T Easy vectors (Promega, Madison, Wisconsin, USA) using T7/Sp6 RNA polymerase (Roche Diagnostics, Basel, Switzerland). Anti-DIG alkaline phosphatase antibody (Roche Diagnostics, Basel, Switzerland) was used for detection, followed by visualization with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) (Roche Diagnostics, Basel, Switzerland). Embryos were imaged using a Leica M205 FA epifluorescence microscope (Leica, Wetzlar, Germany Applications Suite). Primers used for the amplification of stag riboprobes are listed in Supplementary Table 3.

miR-122-3p, miR-122-3p-mutant (miR-122-3p Mut) with disrupted base pairing between LINC00665 and miR-122-3p or its negative control (NC) were purchased from GenePharma (Shanghai, China). miRNAs were biotin-labeled using the Biotin RNA Labeling Mix (Roche, Basel, Switzerland) and T7/SP6 RNA polymerase (Roche). Whole cell lysates were mixed and incubated with biotinylated RNAs. The complexes were then incubated with Streptavidin agarose beads (Invitrogen) for 1 h at 37°C. The beads were washed, and RNA level were analyzed by qRT-PCR [29 ].

Plasmid DNA was linearized with appropriate restriction endonucleases for 5 h at 37 °C, purified using QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany), and the degree of linearization was examined on a 1% agarose gel. In vitro transcription to produce digoxigenin (DIG)-labeled RNA probe was carried out combining linearized plasmid, 1 μg DIG labeling mix (Roche, Mannheim, Germany), 2 μl transcription buffer, 2 μl RNase inhibitor (Roche, Mannheim, Germany), 1 μl T7/Sp6 RNA polymerase (Roche), and 2 μl RNase-free ddH₂O to a final volume of 20 μl. The mix was incubated at 37 °C for 2 h. This was followed by DNase I treatment for 15 min at 37 °C. Labeled RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany); probe length was verified by agarose gel and then dissolved in 150 μl hybridization buffer and stored at − 20 °C until use.

linc00673 was prepared in vitro by transcribing from vector pcDNA3.1-linc00673. RNA biotin-labeling was performed with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN) and T7/SP6 RNA polymerase (Roche Diagnostics, Indianapolis, IN). RNA purification was carried out using through RNeasy Mini Kit (Qiagen, Valencia, CA). Next, 1 milligram A549 cell lysates and 3 μg purified biotinylated transcripts were mixed and incubated with streptavidin agarose beads (Invitrogen) for 1 h at 25°C. Subsequently, the beads were washed and sodium dodecyl sulfate (SDS) buffer added to the eluent. Finally, the standard Western blot technique was used to analyze the retrieved protein.