For the 2′-O-methylation assay, 50 ng of Cy5-labeled primer (Sigma) was annealed to 40 nm RNA by heating to 75 °C for 5 min and snap-cooling on ice. Reverse transcription was carried out using 5 units of AMV reverse transcriptase (Promega) in 20 mm Tris-HCl, pH 7.5, 100 mm KCl, 0.5 mm dNTPs with 0–4 mm MgOAc. For IFIT binding experiments, 25 ng of Cy5-labeled primer was annealed to 10 nm RNA and then incubated with indicated concentrations of IFIT in 20-μl reactions containing 20 mm Tris-HCl, pH 7.5, 100 mm KCl, 2.5 mm MgCl2, 1 mm ATP, 0.2 mm GTP, 1 mm DTT, 0.25 mm spermidine, 0.1 unit/μl RNaseOUT, and 0.5 mg/ml BSA. The reactions were incubated at 37 ˚C for 10 min before addition of 2.5 units of AMV reverse transcriptase (Promega), 4 mm MgCl2, 0.5 mm dNTPs, and labeled primer, either Cy5 (Fig. S8A ) or 32P (PerkinElmer) (Fig. S8D ). Reverse transcription reactions were incubated at 37 ˚C for 30 min and then stopped with 100 mm EDTA and 10% SDS. cDNA products were extracted with UltraPure phenol:chloroform:isoamylalcohol (25:24:1), pH 8, (ThermoFisher) and ethanol-precipitated. Pellets were resuspended in 91% formamide loading dye and boiled for 5 min at 75 °C for PAGE. cDNA products were separated by 6% denaturing PAGE on 35-cm sequencing gels for 30–60 min and then imaged directly on an FLA7000 Typhoon scanner (GE Healthcare).
Amv reverse transcriptase
Manufactured by Promega
Sourced in United States, United Kingdom, China, Germany, Japan
AMV reverse transcriptase is an enzyme used for the synthesis of complementary DNA (cDNA) from RNA templates. It catalyzes the conversion of single-stranded RNA into double-stranded cDNA, which can then be used in various molecular biology applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (83 mentions)
Trizol, by Thermo Fisher Scientific (38 mentions)
Rneasy mini kit, by Qiagen (31 mentions)
Rnasin, by Promega (14 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (13 mentions)
Random primer, by Promega (12 mentions)
Iq sybr green supermix, by Bio-Rad (11 mentions)
Lightcycler 480, by Roche (11 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (10 mentions)
Rneasy kit, by Qiagen (10 mentions)
Taq polymerase, by Promega (9 mentions)
Rnase inhibitor, by Promega (9 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Random hexamer, by Thermo Fisher Scientific (7 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (7 mentions)
Sybr green master mix, by Thermo Fisher Scientific (7 mentions)
Gotaq qpcr master mix, by Promega (6 mentions)
Sybr green master mix, by Bio-Rad (6 mentions)
M mlv reverse transcriptase, by Promega (6 mentions)
Sybr green qpcr kit, by Bio-Rad (5 mentions)
410 protocols using amv reverse transcriptase
1
2'-O-Methylation and IFIT Binding Assays
2
Quantifying Gene Expression via RT-PCR
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At 24 h after the last UVB treatment, total RNA was extracted from the cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and preserved at -80 °C prior to use. Then, 1 μg of RNA was reverse-transcribed into cDNA using the AMV reverse transcriptase (Promega, Madison, WI, USA) in a 20-μL reaction solution that included 4 μL of 5× buffer, 1 μL of oligo-(dT), 1 μL of dNTPs, 0.5 μL of RNase inhibitor, and 0.5 μL of AMV reverse transcriptase with double-distilled water to make up the final volume. The cDNA was amplified using a RT-PCR kit (Takara, Shiga, Japan); the primer sequences are listed in Table 1. The amplification reaction assays contained 1× SYBR Green PCR Mastermix and primers (Applied Biosystems, the Netherlands) at optimal concentrations. The following cycling parameters were used in the StrataGene Mx3000p System (Agilent Technologies, USA): 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s. Melting curves were generated after amplification, and the data were analyzed using the Mxp software. Each sample was tested in triplicate, and three independent experiments were performed.
3
Chloroplast 16S rRNA Methylation Analysis
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Five hundred ng of total RNA, extracted from the indicated genotypes, as described above, was used to perform the primer extension assay. Briefly, the gene-specific primer was designed with the reverse-complementary sequence for the target region (between 963 and 979; 5'-AAGGCACCCCTCTCTTT-3') of the chloroplast 16S rRNA. The primer and DNA marker were end-labeled with 10 µCi of [γ-32 P]ATP by T4 polynucleotide kinase (Promega) and then used for reverse transcription with avian myeloblastosis virus (AMV) reverse transcriptase (Promega) according to the manufacturer's instructions. The products of reverse transcription were separated on 8% denaturing polyacrylamide gel containing 40% polyacrylamide/bis (19:1) solution, 7M urea, and 1× TBE buffer. The gel was washed with a fixation solution [1× TBE buffer, 10% (v/v) ethanol, 10% (v/v) methanol], and then the radioactivity was visualized with a Personal Molecular Imager (PMI) system (Bio-Rad). To quantify the levels of endogenous m 2 G915 in chloroplast 16S rRNA, primer extension products reversetranscribed with a gene-specific primer (reverse-complementary to the 16S rRNA nucleotides 1092-1108; 5'-CAGTCTGTTCAGGGTTC-3') and AMV reverse transcriptase (Promega) were analyzed by qPCR with the primer pairs, as described in Supplementary Figure S4B, and the primer sequences are listed in Supplementary Table S1.
4
Comprehensive RNA Extraction and qPCR Analysis
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TRIzol reagent (Invitrogen, CA, USA) was utilized to extract the entire RNA, and cDNA was generated using AMV reverse transcriptase (Promega, Wisconsin, USA). cDNA was amplified with RR820A SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Osaka, Japan) and a 7500 Fast Real-Time PCR System (Applied Biosystems, MD, USA). The above operations were carried out according to their instructions. The procedures for PCR quantification were performed as previously described (18 (link), 19 (link)). Internal reference was the GAPDH gene. The primers were listed in Supplementary Table 1
5
Colonic Mucosal RNA Extraction and qPCR
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Total RNA from the colonic mucosa of mice was extracted using TRIzol reagent (Invitrogen, USA). First-strand cDNA templates were synthesized using AMV reverse transcriptase (Promega, USA)38 (link) with hexanucleotide random primers. Conventional PCR was carried out in a BioRad thermocycler. Quantitative real-time PCR amplification was carried out using specific primers designed based on cDNA sequences deposited in the GenBank database39 (link) (Table 1 ). Real-time PCR was performed in a Bio-rad CFX-96 Real-Time PCR system using SYBR green PCR reagent kits (Bio-rad). Briefly, each of the 40 cycles consisted of denaturation at 95 °C for 10 s, primer annealing at 55 °C for 20 s, and extension at 72 °C for 30 s after an initial hot start at 95 °C for 30 s, with a final incubation at 72 °C for 10 min. The relative amount of transcripts was calculated using the 2−ΔΔCt formula as the described40 (link), where DCt is the value from the threshold cycle (Ct) value of the treated sample subtracting the Ct value of the untreated or zero time-point control sample. The relative amount of the sample mRNA was normalized to the GAPDH mRNA.
6
RT-qPCR Gene Expression Analysis
7
Quantitative Gene and Protein Analysis
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Total RNA was isolated from cells using TRIzol Reagent (Invitrogen), and then complementary DNA (cDNA) was synthesized using AMV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer's instructions. The cDNA was used as a template for quantitative real-time PCR using an ABI Prism 7500 real-time PCR instrument (Applied Biosystems, Carlsbad, CA, USA). The primers used for real-time quantitative PCR are listed in Supplementary Table 1 .
For western blotting analysis, total protein was prepared from human liver cell lines and clinical hepatocellular carcinoma tissue samples. Immunoblotting was performed according to standard procedures with polyclonal rabbit anti-human LDHA, anti-human c-Myc, anti-α-Tubulin (Cell Signaling, Bedford, MA, USA), monoclonal mouse anti-human NDRG2 (Abnova, Taipei, Taiwan), and polyclonal rabbit anti-human β-actin (Biosynthesis Biotechnology, Beijing, China) antibodies.
For western blotting analysis, total protein was prepared from human liver cell lines and clinical hepatocellular carcinoma tissue samples. Immunoblotting was performed according to standard procedures with polyclonal rabbit anti-human LDHA, anti-human c-Myc, anti-α-Tubulin (Cell Signaling, Bedford, MA, USA), monoclonal mouse anti-human NDRG2 (Abnova, Taipei, Taiwan), and polyclonal rabbit anti-human β-actin (Biosynthesis Biotechnology, Beijing, China) antibodies.
8
Nucleic Acid Extraction and Virus Detection
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For the analyses conducted at Agroscope, nucleic acids were extracted from plant and insect tissues using a 3 % CTAB protocol (Mahillon et al., 2022 (link)). PCR and RT-PCR amplifications were performed using the AMV reverse transcriptase and Taq polymerase (Promega) in combination with primers listed in Table S1 and S2. Reaction mixes and thermocycler conditions have been described before (Mahillon et al., 2022 (link)). For the detection of grapevine viruses and viroids, RT-PCR analyses were conducted as previously described (Kofalvi et al., 1997 (link); De Meyer et al., 2000 ; Jiang et al., 2009 (link); Terlizzi et al., 2011 ; Ghanem-Sabanadzovic et al., 2012 (link); Beuve et al., 2013 ). Amplicons were analyzed by agarose gel electrophoresis.
9
RNA Extraction and RT-PCR Analysis
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Total RNA was isolated from cells using an easy-BLUE kit (Intron, Seoul, South Korea). The RNA was extracted with chloroform and precipitated with isopropanol. The RNA pellet was washed with 75% ethanol and resuspended in DEPC-treated water. Approximately 3 μg of RNA was used to generate cDNA using AMV Reverse Transcriptase (Cat# M5108) and Random Primer (Cat# C1181) (both from Promega US, Madison, WI, USA). Aliquots of the resulting cDNAs were amplified using following primers: rat syndecan-2, 5′-ATGCGGGTACGAGCCACGTC-3′ (forward) and 5′-CGGGAGCAGCACTAGTGAGG-3′ (reverse); human SDC2 5′-ACATCTCCCCTTTGCTAACGGC-3′ (forward) and 5′-TAACTCCATCTCCTTCCCCAGG-3′ (reverse), human MMP-7 5′-GGTCACCTACAGGATCGTATCATAT-3′ (forward) and 5′-CATCACTGCATTAGGATCAGAGGAA-3′ (reverse), human GAPDH 5′-CCACCCATGGCAAATTCCATGGCA-3′ (forward) and 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ (reverse). After an initial denaturation at 94 °C for 5 min, the samples were subjected to 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s. Human GAPDH was amplified as an internal control. The generated PCR products were separated by 1% agarose gel electrophoresis.
10
Bacterial RNA Extraction and RT-PCR Analysis
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Bacteria were harvested by centrifugation and bacterial RNA were stabilized by a 0.1% SDS, 1% acidic phenol, 19% ethanol mixture [67 ]. Total RNA were purified using the RNeasy Mini Kit (Qiagen) and genomic DNA was removed using the TURBO DNA-free kit (Ambion) according to manufacturers’ instructions. Then two micrograms of total RNA were reverse transcribed in presence of random hexamers using the AMV Reverse Transcriptase (Promega). PCR amplification mixtures contained 0.5 μM of each primer (Table S1), 200 μM deoxyribonucleoside triphosphate, 1.5 mM MgCl2, 1 U of GoTaq DNA polymerase (Promega) and 1X Green GoTaq Flexi Buffer (Promega). The temperature cycling for the amplification was performed in a Bio-Rad thermocycler (MyCycler) as follows: 1 cycle at 95°C for 3 min; 30 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 30 s; and finally, 1 cycle at 72°C for 5 min. Reactions without RT were performed to check the absence of DNA contamination in RNA samples. All RT-PCR experiments were duplicated from at least two independent bacterial cultures and RNA extractions.
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