FAW larvae were collected from the corn field in Baoting, Hainan, Southern China (19°14′45.12″N, 109°89′40.40″E) on May 5th, 2019. We established a laboratory colony by feeding the larvae on artificial diet under conditions of 25 ± 1 °C, 14 h:10 h (L:D) photoperiod, and 55 ± 5% relative humidity. The third instar larvae were individually transferred into a small box and reared until pupation. Adults were transferred into a home-made insect raise cage (Patent no. 201921652702.2) after inclusion and fed with 10% hydromel (v/v). Once adequate fourth instar larvae were obtained, we rinsed them in 75% ethanol (v/v) and sterile phosphate-buffered saline (PBS, pH 7.2) twice, respectively. Next, the brain was dissected in PBS containing 1 unit/µL Murine RNase inhibitor (Vazyme, Nanjing, China) under an Olympus SZ61 stereomicroscope (Olympus, Tokyo, Japan). We collected the sample into a 1.5 ml Eppendorf tube for subsequent RNA extraction.
Murine rnase inhibitor
Manufactured by Vazyme
Sourced in China
Murine RNase inhibitor is a laboratory reagent designed to inhibit RNase activity in murine (mouse) biological samples. It functions by binding to and deactivating RNase enzymes, which helps preserve the integrity of RNA molecules during sample processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sz61 stereomicroscope, by Olympus (1 mentions)
Pyrophosphatase, by Vazyme (1 mentions)
Site directed mutagenesis kit, by Vazyme (1 mentions)
Dsrna quantitative kit, by Vazyme (1 mentions)
Mz 16a stereomicroscope, by Leica (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qubit rna assay kit in qubit 2.0 flurometer, by Thermo Fisher Scientific (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Nanophotometer spectrophotometer, by Implen (1 mentions)
Splintr ligase, by New England Biolabs (1 mentions)
Tris hcl ph7, by Thermo Fisher Scientific (1 mentions)
Malachite green oxalate, by Merck Group (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Rnase a, by Tiangen Biotech (1 mentions)
Nebuffer 1, by Roche (1 mentions)
Murine rnase inhibitor, by Roche (1 mentions)
Nebuffer 2, by New England Biolabs (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Champagne taq dna polymerase, by Vazyme (1 mentions)
7 protocols using murine rnase inhibitor
1
Isolation and RNA Extraction from FAW Larval Brains
2
T7-based Heterologous Protein Expression
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The pQE-30 plasmid and Escherichia coli BL21(DE3) were purchased from Novagen Inc. (Madison, WI, USA) for T7 RNAP heterologous expression. The site-directed mutagenesis kit and dsRNA quantitative kit were supplied by Vazyme Co., Ltd. (Nanjing, China). ATP, GTP, CTP, UTP, pyrophosphatase, and murine RNase inhibitor were purchased from Vazyme Co., Ltd. (Nanjing, China). MgCl2, glycerol, dithiothreitol (DTT), and other chemicals were purchased from Aladdin Industrial Inc. (Shanghai, China) and other commercial sources.
3
Transcriptome Analysis of Wasp Venom Glands
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Mated female wasps aged 2–5 days were anesthetized at 4°C for 10 min, rinsed in 75% ethanol (v/v) once, and then rinsed in sterile phosphate-buffered saline (PBS, pH 7.2) thrice. Subsequently, the females were dissected in PBS containing 1 unit/μL Murine RNase inhibitor (Vazyme, Nanjing, China) on an ice plate under a Leica MZ 16A stereomicroscope (Leica, Wetzlar, Germany), the venom apparatus (venom reservoirs and associated glands, henceforth, called the VG) and carcasses (the female body minus venom apparatus, henceforth, called the CA) were collected into 1 mL TRIzol reagent (Invitrogen, Carlsbad, CA, United States), respectively. Total RNA was extracted according to the manufacturer’s protocol. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, United States). RNA concentration was measured using the Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, United States). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, United States).
4
Oligonucleotide-based protocol synthesis
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All the oligonucleotide probes, blocker, and target sequences we used were synthesized at GENEWIZ Inc. (Suzhou, China) and these sequences were listed in Supplementary Tables S1 and S2 . Splint R ligase and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA, USA). Murine RNase Inhibitor was acquired from Vazyme Biotech (Nanjing, China). Malachite green oxalate was purchased from Sigma–Aldrich (Darmstadt, Germany). T7 RNA polymerase was purchased from TaKaRa Biomedical Technology Co., Ltd (Beijing, China). NTPs was obtained from Sangon Biotech. (Shanghai, China). Tris-HCL (pH 7.5) was acquired from Thermo Fisher Scientific (Waltham, Massachusetts, USA). And magnesium chloride solution was obtained from J&K Scientific Ltd. (Beijing, China). ATP was purchased from MDBio, Inc (Taiwan, China).
5
SARS-CoV-2 Pseudovirus Detection Assay
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Single-walled carbon nanotubes (SCNTs, 15 mg/mL), multi-walled carbon nanotubes (MCNTs, 1 mg/mL), and graphene oxide (GO, 1 mg/mL) were purchased from XianFeng Nanotechnology Co., Ltd. (Nanjing, China). Magnetic beads (MBs; BeaverBeads™ NH2, 500 nm, 10 mg/mL) were obtained from Beaver Co., Ltd. (Suzhou, China). RNase A (10 mg/mL) was purchased from TianGen biotech Co., Ltd. (Beijing, China). Murine RNase inhibitor was obtained from Vazyme Biotech Co., Ltd. (Nanjing, China). SARS-CoV-2-ab II EMN pseudovirions (1.25 × 1010 copies/mL), influenza A H1N1-M1 pseudovirions (1.7 × 1010 copies/mL), and influenza B NS1 pseudovirions (7.1 × 109 copies/mL) were all purchased from FuBio Co., Ltd. (Suzhou, China). TransScript® One-Step RT-qPCR SuperMix kit was obtained from TransGen Biotech Co., Ltd. (Beijing, China), and viral transport media was supplied by Navid Biotech Co., Ltd. (Qingdao, China). All oligonucleotides used in this study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals were analytical grade.
6
Orthogonal Cas12b and Cas13a Assays
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For the dsDNA target cleavage assay of AapCas12b, 1.2 μM AapCas12b protein, 1.2 μM sgRNA, 300 ng dsDNA substrate and 0.8 unit/μL Murine RNase Inhibitor (Vazyme, China) were incubated for 1 h at 60°C in cleavage buffer (NEBuffer 2.1, NEB, USA) as discribed.54 (link)
For the ssRNA target cleavage assay of LwaCas13a, 0.6 μM LwaCas13a protein, 0.6 μM crRNA, 1300 ng ssRNA substrate and 0.8 unit/μL Murine RNase Inhibitor were incubated for 1 h at 37°C in NEBuffer 1.1 as discribed.97 (link)
To test the orthogonal collateral cleavage of AapCas12b and LwaCas13a, 50 nM AapCas12b protein, 150 nM sgRNA, 10 nM dsDNA substrate, 0.8 unit/μL Murine RNase Inhibitor, 500 nM quenched ssDNA fluorescent probes and 500 nM quenched ssRNA fluorescent probes were incubated in NEBuffer 1.1 for 15 min at 60°C as discribed.50 For LwaCas13a, an assay was performed with 50 nM LwaCas13a protein, 50 nM crRNA, 10 nM ssRNA substrate, 0.8 unit/μL Murine RNase Inhibitor, 500 nM quenched ssDNA fluorescent probes, 500 nM quenched ssRNA fluorescent probes and NEBuffer 1.1 for 15 min at 37°C as discribed.50 The fluorescence signals of these reactions were recorded every minute by a LightCycler 96 (Roche, Swiss) in the FAM channel (for ssDNA probe) and ROX channel (for ssRNA probe). Detailed probe sequences are listed inTable S3 .
For the ssRNA target cleavage assay of LwaCas13a, 0.6 μM LwaCas13a protein, 0.6 μM crRNA, 1300 ng ssRNA substrate and 0.8 unit/μL Murine RNase Inhibitor were incubated for 1 h at 37°C in NEBuffer 1.1 as discribed.97 (link)
To test the orthogonal collateral cleavage of AapCas12b and LwaCas13a, 50 nM AapCas12b protein, 150 nM sgRNA, 10 nM dsDNA substrate, 0.8 unit/μL Murine RNase Inhibitor, 500 nM quenched ssDNA fluorescent probes and 500 nM quenched ssRNA fluorescent probes were incubated in NEBuffer 1.1 for 15 min at 60°C as discribed.50 For LwaCas13a, an assay was performed with 50 nM LwaCas13a protein, 50 nM crRNA, 10 nM ssRNA substrate, 0.8 unit/μL Murine RNase Inhibitor, 500 nM quenched ssDNA fluorescent probes, 500 nM quenched ssRNA fluorescent probes and NEBuffer 1.1 for 15 min at 37°C as discribed.50 The fluorescence signals of these reactions were recorded every minute by a LightCycler 96 (Roche, Swiss) in the FAM channel (for ssDNA probe) and ROX channel (for ssRNA probe). Detailed probe sequences are listed in
7
One-step RT-qPCR Assay for Gene Expression
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The one-step RT-qPCR assay (referred to as our RT-qPCR assay) was performed in 96-well plates with a reaction volume of 30 μL. For the assay, 3 μL of 10× Taq Buffer, 0.24 μL of Champagne Taq DNA Polymerase, 0.35 μL of HiScript II Reverse Transcriptase, 0.25 μL of Murine RNase inhibitor, 0.3 μL of heat-labile UDG, 2.4 μL of MgCl2 and 0.6 μL of dNTP mix (Vazyme Biotech Co., Ltd., Nanjing, China), 0.6 μL of forward primers and 0.6 μL of reverse primers for each gene, 0.15 μL of probes for each gene, 13.81 μL of nuclease-free water, and 5 μL of template were mixed per reaction. The cycling conditions for each assay consisted of incubating the template at 55 °C for 5 min and 95 °C for 30 s, followed by 45 cycles of RT-qPCR, with each cycle consisting of 95 °C for 5 s and then 62 °C for 20 s. All reactions were carried out with a SLAN®-96S Real-Time PCR System (Hongshi Medical Technology Co., Ltd., Shanghai, China).
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