Reverse transcription reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Reverse Transcription Reagents are a set of laboratory chemicals and enzymes used for the conversion of RNA into complementary DNA (cDNA). This process is known as reverse transcription and is a crucial step in various molecular biology applications, such as gene expression analysis and the detection of RNA viruses.

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93 protocols using reverse transcription reagent

Quantitative Gene Expression Analysis

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Total RNA was extracted using Trizol (Thermo-Fisher-Scientific) following the manufacturer’s instructions. 1 μg of total RNA was used to synthesize cDNA using reverse transcription reagents (Thermo-Fisher-Scientific) after pre-treatment with DNAseI (Thermo-Fisher-Scientific) to avoid contamination from genomic DNA. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using TaqMan® Brilliant II QPCR Master Mix (Agilent Technologies, CA, USA) on a Stratagene-MX3005P (Agilent-Technologies) under standard conditions. The housekeeping gene glucoronidase β gene (GUSB) was used as an internal reference.^{12 (link)} TaqMan® Gene Expression Assays (Thermo-Fisher-Scientific) were used (Online Supplementary Table S1).

Magistroni V., Mauri M., D’Aliberti D., Mezzatesta C., Crespiatico I., Nava M., Fontana D., Sharma N., Parker W., Schreiber A., Yeung D., Pirola A., Readelli S., Massimino L., Wang P., Khandelwal P., Citterio S., Viltadi M., Bombelli S., Rigolio R., Perego R., Boultwood J., Morotti A., Saglio G., Kim D.W., Branford S., Gambacorti-Passerini C, & Piazza R. (2019). De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways. Haematologica, 104(9), 1789-1797.

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Transcriptome Analysis of Vancomycin-Treated Cells

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Cells were grown up to OD₆₀₀ = 0.5 and treated with 1 μg ml^-1 vancomycin. After 10 min of incubation, 5 ml of sample were collected, and RNA was isolated using the RNeasy Kit (Qiagen) following manufacturer’s instructions. The RNA was treated with Turbo DNase, and then purified by phenol-chloroform extraction. The RNA was quantified using a Nanodrop, its purity assessed by the 260/280 ratio, and integrity monitored by agarose gel electrophoresis. A total of 1 μg of RNA was reverse transcribed using the P46 primer and the Reverse Transcription Reagents (Thermo Fisher Scientific, US) following manufacturer’s instructions. The cDNA was column purified and then treated with the terminal transferase enzyme using CTP to add a homopolymeric cytosine tail at the 3’ end. Then, the cDNA was PCR amplified using the abridged anchor primer (AAP) and DR288 primers by using a touchdown PCR followed by a conventional PCR. The PCR product was verified by electrophoresis and sequenced using the DR289 primer.

Rojas-Tapias D.F, & Helmann J.D. (2018). Stabilization of Bacillus subtilis Spx under cell wall stress requires the anti-adaptor protein YirB. PLoS Genetics, 14(7), e1007531.

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TGF-β1 and RhoA Inhibitor Assay

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All reagents were purchased from Sigma-Aldrich (Poole, UK) or Thermo Fisher Scientific (Paisley, UK) unless otherwise stated. Reverse-transcription reagents, siRNA transfection reagents, and real-time quantitative PCR (qPCR) primers and reagents were purchased from Thermo Fisher Scientific. Other reagents used were recombinant human TGF-β1 (R&D Systems, Abingdon, UK) and the RhoA inhibitor Rhosin (G04; Merck Millipore, Watford, UK).

Midgley A.C., Woods E.L., Jenkins R.H., Brown C., Khalid U., Chavez R., Hascall V., Steadman R., Phillips A.O, & Meran S. (2020). Hyaluronidase-2 Regulates RhoA Signaling, Myofibroblast Contractility, and Other Key Profibrotic Myofibroblast Functions. The American Journal of Pathology, 190(6), 1236-1255.

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Quantifying RNA Expression by RT-qPCR

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Five million cells were lysed in TRIzol (Thermo Fisher Scientific) and total RNA was extracted according to manufacturer’s instructions. 1 µg of total RNA was used to synthesize cDNA using reverse transcription reagents (Thermo Fisher Scientific) after pre-treatment with DNAseI (Thermo Fisher Scientific) to avoid contamination from genomic DNA. Real-Time-Quantitative PCR (RT-qPCR) was performed using Luna^® Universal Probe qPCR Master Mix (NEB) on a Stratagene-MX3005P (Agilent Technologies) under standard conditions. The housekeeping glucoronidase beta gene (GUSB) was used as an internal reference. TaqMan^® Gene Expression Assays Hs01071698_m1 (Thermo Fisher Scientific) was used.

Fontana D., Mauri M., Renso R., Docci M., Crespiatico I., Røst L.M., Jang M., Niro A., D’Aliberti D., Massimino L., Bertagna M., Zambrotta G., Bossi M., Citterio S., Crescenzi B., Fanelli F., Cassina V., Corti R., Salerno D., Nardo L., Chinello C., Mantegazza F., Mecucci C., Magni F., Cavaletti G., Bruheim P., Rea D., Larsen S., Gambacorti-Passerini C, & Piazza R. (2020). ETNK1 mutations induce a mutator phenotype that can be reverted with phosphoethanolamine. Nature Communications, 11, 5938.

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Quantifying DHRS4-AS1 and SOCS5 in HCC

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Human HCC tissues and adjacent normal tissues were grant for further RNA extraction. Tissues homogenate as well as normal hepatic cells (L02) and HCC cells (Hep3B, YY-8103, Focus, HCCLM3 and Huh7) were added 1 ml of Trizol reagent (Invitrogen, Carlsbad, CA, USA) to lyse cells. The cDNA synthesis was conducted using reverse transcription reagents (Thermo Scientific, Waltham, MA, USA). Chloroform was then added to the mixture at ambient temperature, which was followed by the addition of isopropanol. Ultimately, the suspension was centrifugated to obtain RNA from the sediments. The expression of DHRS4-AS1 and SOCS5 were determined by qRT-PCR by utilizing the PrimeScript RT reagent Kit and SYBR Prime Script RT-PCR Kits (TaKaRa Bio, Inc., Shiga, Japan) according to the manufactures’ guidance, and GAPDH as the internal control. The expression of miR-522-3p was measured using TaqMan MicroRNA Assays and U6 as the internal control. 2^−ΔΔct were used as the calculating method for the results data. The primer sequences have been provided in Table 1.

Zhou Y., Li K., Zou X., Hua Z., Wang H., Bian W., Wang H., Chen F, & Dai T. (2021). LncRNA DHRS4-AS1 ameliorates hepatocellular carcinoma by suppressing proliferation and promoting apoptosis via miR-522-3p/SOCS5 axis. Bioengineered, 12(2), 10862-10877.

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RNA Extraction and qRT-PCR Analysis

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Lung tissues and RAW264.7 cells were homogenized in RNAiso plus (Takara, Shiga, Japan) lysis buffer, and total RNA was extracted. RNAs were reverse-transcribed using reverse transcription reagents (Thermo Scientific, USA). qRT-PCR was performed using SYBR Green Master Mix on LightCycler 480 sequence-detector system. All the primers in this study were obtained from Sangon Biotech (Shanghai, China). β-actin or GAPDH was used as the internal control. The comparative Ct method (2^{− ΔΔCt}) was utilized to analyze data.

Chen M., Lv J., Guo N., Ji T., Fang Y., Wang Z, & He X. (2024). Crtc1 deficiency protects against sepsis-associated acute lung injury through activating akt signaling pathway. Journal of Inflammation (London, England), 21, 12.

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Investigating EMT Regulation in Colorectal Cancer

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Human colorectal cancer tissues and colorectal cell lines were collected to extract the RNA for continual experiment. In order to lyse cells and avoid RNA lysis, 1 mL Trizol reagent (Invitrogen, Carlsbad, CA, USA) was added to cell and tissue suspension. Reverse transcription reagents (Thermo Scientific, Waltham, MA, USA) were utilized to completed cDNA synthesis. Additional chloroform at room temperature was added to the mixture, then added isopropanol to centrifugate to get RNA precipitate. Real-time qPCR of SERPINE1, miR-148-3p and EMT-related transcripts was conducted through an ABI ViiA 7 sequence detection system (ABI, Carlsbad, CA, USA) and a SYBR Premix Ex Taq™ II kit (TaKaRa Bio, Inc., Shiga, Japan) based on the manufacturer's instruction. The transcription level was calculated by 2^−ΔΔCt method.18 (link)

Hu B., Chen Z., Wang X., Chen F., Song Z, & Cao C. (2021). MicroRNA-148a-3p Directly Targets SERPINE1 to Suppress EMT-Mediated Colon Adenocarcinoma Progression. Cancer Management and Research, 13, 6349-6362.

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Measuring Immunoglobulin Levels and Gene Expression

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We purchased ELISA kits for IgA, IgE, and IgG2a from Huijia Biotechnology Co., Ltd. (Xiamen, China).
Fluorescein isothiocyanate-labeled 4.4 kDa dextran (FD4) was purchased from Sigma-Aldrich (St. Louis, MO). TRIzol reagent was purchased from Sangon Biotech Co., Ltd. (Shanghai, China) . Reverse transcription reagents were purchased from Thermo Fisher Scientific Inc. (Waltham, MA), and primers for quantitative real-time polymerase chain reaction (qRT-PCR) were synthesized by GENEWIZ Biotech Co., Ltd. (Suzhou, China) . Primary antibodies against IgA were purchased from Abcam Co., Ltd. (Cambridge, UK). All other analytical grade solvents and reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) .

Qi C., Ding M., Li S., Zhou Q., Li D., Yu R, & Sun J. (2021). Sex-dependent modulation of immune development in mice by secretory IgA-coated Lactobacillus reuteri isolated from breast milk. Journal of dairy science, 104(4).

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Quantitative PCR of Key Transcripts

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Total RNA was extracted with Trizol reagent following standard protocol (Thermo Fisher Scientific). One microgram of RNA was used to synthesize cDNA using Reverse Transcription Reagents (Thermo Fisher Scientific) after pretreatment with DNAseI to avoid contamination from gDNA. Real-time Q-PCR was performed using TaqMan® Brilliant II QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA) on a Stratagene-MX3005P (Agilent Technologies, Santa Clara, USA) under standard conditions. The housekeeping gene GUSB was used as an internal reference^{43 (link)}. TaqMan® Gene Expression Assays (Thermo Fisher Scientific) were used: SKIDA1 (Hs01096520), NFE2L2 (Hs00975961), PDE4D (Hs03988495), FBXO8 (Hs00942619), CEP44 (Hs00604612), COBLL1 (Hs01117513), BMP5 (Hs00234930), ERBB4 (Hs00955525), CDKN1B (Hs01597588), SETBP1 (Hs00210203), RUNX1 (Hs01021971), MECOM (Hs00602795).

Piazza R., Magistroni V., Redaelli S., Mauri M., Massimino L., Sessa A., Peronaci M., Lalowski M., Soliymani R., Mezzatesta C., Pirola A., Banfi F., Rubio A., Rea D., Stagno F., Usala E., Martino B., Campiotti L., Merli M., Passamonti F., Onida F., Morotti A., Pavesi F., Bregni M., Broccoli V., Baumann M, & Gambacorti-Passerini C. (2018). SETBP1 induces transcription of a network of development genes by acting as an epigenetic hub. Nature Communications, 9, 2192.

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Quantitative RT-PCR analysis of gene expression

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Total RNA from cells and kidney tissues was extracted with TRIzol reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer’s instruction. Complementary DNA was synthesized using reverse transcription reagents (Thermo Fisher Scientific, N8080234). Quantitative real-time PCR was performed with the TB Green Premix Ex Taq II reagent (TaKaRa, RR820B) on LightCycler96 Real-Time PCR System (Roche Life Science). For quantification, the mRNA levels of target genes were normalized to the mRNA levels of Gapdh. Primers used were as follows: Bnip3—forward, 5′-GCTCCCAGACACCACAAGAT-3′ and reverse, 5′-TGAGAGTAGCTGTGCGCTTC-3′; Tnf-α—forward, 5′-CAGGCGGTGCCTATGTCTC-3′ and reverse, 5′-CGATCACCCCGAAGTTCAGTAG-3′; Il-1b—forward, 5′-GAAATGCCACCTTTTGACAGTG-3′ and reverse, 5′-CTGGATGCTCTCATCAGGACA-3′; Gapdh—forward, 5′-AGGTCGGTGTGAACGGATTTG-3′ and Gapdh reverse, 5′-GGGGTCGTTGATGGCAACA-3′.

Tang C., Han H., Liu Z., Liu Y., Yin L., Cai J., He L., Liu Y., Chen G., Zhang Z., Yin X.M, & Dong Z. (2019). Activation of BNIP3-mediated mitophagy protects against renal ischemia–reperfusion injury. Cell Death & Disease, 10(9), 677.

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