Amv reverse transcriptase xl

Manufactured by Takara Bio

Sourced in Japan, United States

AMV Reverse Transcriptase XL is a recombinant reverse transcriptase enzyme derived from Avian Myeloblastosis Virus. It is used for the synthesis of first-strand cDNA from RNA templates.

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36 protocols using amv reverse transcriptase xl

Quantitative RNA Expression Analysis

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Extraction of total RNA from tissues and cells was performed using Ribozol reagent (Invitrogen, USA). AMV Reverse Transcriptase XL (Clontech, USA) and QuantiTect SYBR Green PCR Kit (Qiagen, Shanghai, China) were used to prepare reverse transcription and qPCR reaction, respectively. With 18S rRNA as endogenous control, the expression level of PLAC2 was normalized. With GAPDH as endogenous control, PTEN expression level (a target of miR-21) was normalized.
To detect miRNA-21, mirVana miRNA Isolation Kit (Thermo Fisher Scientific), qScript microRNA cDNA Synthesis Kit (Quantabio, USA) and miScript SYBR Green PCR Kit (QIAGEN, Germany) were used to perform miRNA extractions, miRNA reverse transcriptions and qPCR reactions, respectively. U6 was used as the endogenous control of miRNA-21. Three replicates were set for each experiment, and 2^-ΔΔCT method was used to process data.

Xia H., Xiu M., Gao J, & Jing H. (2019). LncRNA PLAC 2 downregulated miR-21 in non-small cell lung cancer and predicted survival. BMC Pulmonary Medicine, 19, 172.

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Quantifying TP73-AS1 and miR-139-3p Expression

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Ribozol (Thermo Fisher Scientific., lnc.) was used to extract total RNAs from tissue specimens as well as WERI-Rb-1 and Y79 cells. Following cDNA synthesis using AMV Reverse Transcriptase XL (Clontech, U.S.A.), qPCR reaction systems were prepared using SYBR Green Master Mix (Bio-Rad, U.S.A.) to detect the expression of TP73-AS1. MiRNA extractions were extracted from tissue specimens as well as WERI-Rb-1 and Y79 cells using mirVana miRNA Isolation Kit (Thermo Fisher Scientific., lnc.). Following reverse transcriptions using TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific., lnc.), qPCR reaction systems were prepared using Applied Biosystems™ TaqMan™ MicroRNA Assay (Thermo Fisher Scientific) to detect the expression of miR-139-3p with U6 as endogenous control. All qPCR reactions were performed three times and data were processed using 2^−ΔΔC_T method. The sample with the lowest ΔC_T value was set to “1” and all other samples were normalized to this sample.

Xia Z., Yang X., Wu S., Feng Z., Qu L., Chen X., Liu L, & Ma Y. (2019). LncRNA TP73-AS1 down-regulates miR-139-3p to promote retinoblastoma cell proliferation. Bioscience Reports, 39(5), BSR20190475.

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Quantifying FOXD2-AS1 and miR-31 Expression

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Ribozol was used to extract total RNAs from RB tissues and cells. In brief, the reverse transcription was performed to synthesize the first-strand cDNA chain using AMV Reverse Transcriptase XL (Clontech, USA). To detect the expression of FOXD2-AS1, qPCR was carried out using SYBR Green Master Mix (Bio-Rad, USA) on an ABI 7500 System. All experiments were performed 3 times, and the data were processed using the 2^−ΔΔCT method. qRT-PCR primers were as follows: FOXD2-AS1 forward: 5′-TGGACCTAGCTGCAGCTCCA-3′ and reverse: 5′-AGTTGAAGGTGCACACACTG-3′; PAX9 forward: 5′-ACCACATTTACTCATATCCCAGTCCCA-3 and reverse: 5′-GGCTCCCTTCTCCAATCCATTCA-3′; GAPDH forward: 5′-TATGATGATATCAAGAGGGTAGT-3′ and reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′; miR-31 forward: 5′-ACGCGGCAAGATGCTGGCA-3′ and reverse: 5′-CAGTGCTGGGTCCGAGTGA-3′; and U6 forward: 5′-CTCGCTTCGGCAGCACA-3′ and reverse: 5′-AACGCTTCACGAATTTGCGT-3′.

Liang Y., Wang H., Song R, & Yin X. (2022). lncRNA FOXD2-AS1 Promotes the Retinoblastoma Cell Viability and Migration by Sponging miR-31. BioMed Research International, 2022, 7723425.

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Quantifying PLAC2 and PTEN Expression

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Ribozol (Thermo Fisher Scientific., lnc.) was mixed with Y79 and WERI-Rb-1 cells (1 ml per 10⁵ cells) and tissues (0.5 ml per 0.02 g tissue) to extract total RNAs. All RNA samples were digested with DNase I. AMV Reverse Transcriptase XL (Clontech Laboratories, Inc.) was used to perform reverse transcription by incubating at 25°C for 10 min, 55°C for 20 min and 80°C for 10 min. SYBR Green Master Mix (Bio-Rad Laboratories, Inc.) was used to prepare qPCR reaction mixtures. The expression of PLAC2 and PTEN was detected using 18S rRNA or GAPDH as endogenous control, respectively. Reaction conditions were: 95°C for 1 min, followed by 40 cycles of 95°C for 10 sec and 60°C for the 50 sec. It is worth noting that multiple primers were used and similar results were obtained. Primer sequences were: 5′-CGGCTACTAGCGGTTTTAC-3′ (forward) and 5′-AAGAAGATGCGGCTGACTG-3′ (reverse) for GAPDH; 5′-TGTGGCCCAAACTCAGGGATACA-3′ (forward) and 5′-GATGACAGTGGCTGGAGTTGTC-3′ for PLAC2 (reverse); 5′-GAGTTCCCTCAGCCGTTACCT-3′ (forward) and 5′-AGGTTTCCTCTGGTCCTGGTA-3′ for (reverse) PTEN mRNA; 5′-GCTTAATTTGACTCAACACGGG-3′ (forward) and 5′-GCTATCAATCTGTCAATCCTGTC-3′ for (reverse) 18S rRNA. All experiments were repeated 3 times and data were processed using the 2^−ΔΔCq method (12 (link)). The sample with the highest ΔCq value was set to ‘1’, all other samples were normalized to this sample.

Song L., Qi Y, & Lin M. (2020). Long noncoding RNA PLAC2 regulates PTEN in retinoblastoma and participates in the regulation of cancer cell apoptosis. Oncology Letters, 19(3), 2489-2494.

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Quantitative Analysis of ELF3-AS1 and GLUT1

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Ribozol reagent (VWR Life Science, USA) was mixed with 10⁵ cells or 0.03 g tissue (ground in liquid nitrogen before use) to perform total RNA extractions. Following DNase I digestion, AMV Reverse Transcriptase XL (Clontech, USA) and Luna® Universal One-Step RT-qPCR Kit (NEB, USA) were used to perform reverse transcriptions and prepare qPCR reaction mixtures. With 18S rRNA or GAPDH as endogenous control, expressions of ELF3-AS1 and GLUT1 mRNA were detected and expression levels were normalized based on g2^−∆∆CT method. Primer sequences were as follows: 5ʹ-TGAAGTCATCACGAACCGC-3ʹ (forward) and 5ʹ-GGAGCCCCAAGTTAATGCG-3ʹ (reverse) for ELF3-AS1; 5ʹ-AGGTGATCGAGGAGTTCTA-3ʹ (forward) and 5ʹ-TCAAAGGACTTGCCCAGTTT-3ʹ (reverse) for GLUT1; 5ʹ-CCAGGGCTGCTTTTAACTCT-3ʹ (forward) and 5ʹ-GGACTC CACGACGTACTCA-3ʹ (reverse) for GAPDH; 5ʹ-CTACCACATCCAAGGAAGCA-3ʹ (forward) and 5ʹ-TTTTTCGTCACTACCTCCCCG-3ʹ (reverse) for human 18S rRNA. Three replicates were set for each experiment.

Chu H., Li Z., Gan Z., Yang Z., Wu Z, & Rong M. (2019). LncRNA ELF3-AS1 is involved in the regulation of oral squamous cell carcinoma cell proliferation by reprogramming glucose metabolism. OncoTargets and therapy, 12, 6857-6863.

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RNA Extraction and Expression Analysis

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H1581 and H1993 cells (collected at 24 h post-transfection) were mixed with RNAzol reagent (Sigma-Aldrich, USA) with a ratio of 1 mL RNAzol reagent per 10⁵ cells. Tissues were ground in liquid nitrogen and 0.05 g tissue was mixed with 1 mL RNAzol reagent to extract total RNA. All RNA samples were subjected to DNase I digestion. Following reverse transcriptions performed using AMV Reverse Transcriptase XL (Clontech, USA), qPCR mixtures were prepared using the SYBR^® Green master mix (Bio-Rad, USA). The expression of MIR503HG and cyclin D1 mRNA was detected using 18S rRNA and GAPDH as endogenous control. All data were processed using 2^−ΔΔCT method.

Xu S., Zhai S., Du T, & Li Z. (2020). LncRNA MIR503HG Inhibits Non-Small Cell Lung Cancer Cell Proliferation by Inducing Cell Cycle Arrest Through the Downregulation of Cyclin D1. Cancer Management and Research, 12, 1641-1647.

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Quantification of CSFV genomic RNA

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Total RNA was extracted from CSFV-infected PK-15 cells or porcine organs using TRIzol reagent (catalog no. 15596026; Invitrogen). The isolated RNA was collected and reverse transcribed into cDNA with avian myeloblastosis virus (AMV) reverse transcriptase XL (catalog no. 2621; TaKaRa) according to the manufacturer's instructions. Genomic RNA copies of CSFV were quantified using a previously described quantitative real-time reverse transcription-PCR (qRT-PCR) assay (30 (link)).

Li L.F., Yu J., Li Y., Wang J., Li S., Zhang L., Xia S.L., Yang Q., Wang X., Yu S., Luo Y., Sun Y., Zhu Y., Munir M, & Qiu H.J. (2016). Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity. Journal of Virology, 90(9), 4412-4426.

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RT-qPCR for EV71 Viral RNA Quantification

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RNA preparation and RT-qPCR followed the protocol previously described [18 (link)]. The total cellular and viral RNA was extracted using a TRIzol reagent (ThermoFisher Inc., Carlsbad, CA, USA). The reverse transcription (RT) reaction and real-time PCR were performed using an AMV Reverse Transcriptase XL (Takara, Kusatsu, Japan) and the FastStart Universal SYBR Green Master kit (Roche Applied Science, Penzberg, Germany), respectively, according to the manufacturer’s instructions. PCR primer pairs for the VP1 region of the EV71 (BrCr strain) genome were: forward primer: 5′-AGTATGATTGAGACTCGGTG-3′ and reverse primer 5′-GCGACAAAAGTGAACTCTGC-3′ [21 (link)]. PCR primers used for the human β-actin were: forward primer: 5′-AGCCTCGCCTTTGCCGA-3′ and reverse primer 5′-CTGGTGCCTGGGGCG-3′ [22 (link)]. Relative viral RNA level (%) was calculated using the viral cDNA level normalized against that of the human β-actin, and compared with that of the DMSO control, as described previously [22 (link)].

Tseng K.C., Hsu B.Y., Ling P., Lu W.W., Lin C.W, & Kung S.H. (2022). Antidepressant Sertraline Is a Broad-Spectrum Inhibitor of Enteroviruses Targeting Viral Entry through Neutralization of Endolysosomal Acidification. Viruses, 14(1), 109.

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Quantification of HEV and RIG-1 Expressions

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Total RNA was isolated from cells by Trizol (Invitrogn, America) in accordance with the manufacturer's instructions. cDNA was prepared by using AMV Reverse Transcriptase XL (Takara, Japan) in accordance with the provided directions. The copy number of HEV was quantified through SYBR green-based qRT-PCR with HEV-specific primers as described in our previous study [21 (link)]. The expression levels of RIG-1 were quantified by using specific primers described in previous studies [28 (link)]. GAPDH was applied as the housekeeping control. Relative gene expression was calculated through the 2^{−(△Ct of gene − △Ct of GAPDH)} method, where Ct is the threshold cycle. qRT-PCR was performed with a Bio-Rad CFX96TM Real-Time PCR System.

Qian Z., Cong C., Li Y., Bi Y., He Q., Li T., Xia Y., Xu L., Mickael H.K., Yu W., Liu J., Wei D, & Huang F. (2023). Quantification of host proteomic responses to genotype 4 hepatitis E virus replication facilitated by pregnancy serum. Virology Journal, 20, 111.

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Nuclear RNA Extraction and Analysis

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To extract RNA in the nuclear fraction, cells were trypsinized, washed with PBS, and extracted with 250 µl of NP-40 buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40, 40 U/µl RNasin (Promega), and 1 mM dithiothreitol) on ice for 10 min. Nuclei were pelleted by centrifugation at 500× g for 5 min at 4°C, washed once with NP-40 buffer, and processed as in total RNA extraction. Total RNA was extracted using an RNeasy Mini kit (Qiagen) with on-column DNase I digestion following the manufacturer's protocol. 1 or 2 µg of RNA was reverse-transcribed with random nonamers or a telomere-specific 5′-(CCCTAA)₅-3′ primer at 55°C using AMV reverse-transcriptase XL (TaKaRa). cDNA was subjected to either real-time PCR as described for ChIP analyses, or PCR using LA-taq DNA polymerase (TaKaRa).

Wakai M., Abe S., Kazuki Y., Oshimura M, & Ishikawa F. (2014). A Human Artificial Chromosome Recapitulates the Metabolism of Native Telomeres in Mammalian Cells. PLoS ONE, 9(2), e88530.

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