Tetro cdna synthesis kit

Manufactured by Meridian Bioscience

Sourced in United Kingdom, United States, Australia, Germany

The Tetro cDNA Synthesis Kit is a laboratory product that enables the reverse transcription of RNA into complementary DNA (cDNA). The kit provides the necessary reagents and enzymes to facilitate this process, which is a fundamental step in various molecular biology applications.

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Tetro cDNA synthesis Tetro cDNA kit CDNA synthesis kit Tetro kit

358 protocols using tetro cdna synthesis kit

Influenza Virus RNA Extraction and Quantification

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Following perfusion, mouse lung tissue was collected and submerged in RNAlater (Ambion) for 24 hours prior to long term storage at -80°C. RNA was prepared from mouse lungs using Trisure (Bioline) according to the manufacturer’s instructions (Bioline). Total RNA (2 μg) was reverse transcribed using the Tetro cDNA synthesis Kit with random primers according to the manufacturer’s instructions (Bioline). Data are expressed as fold increases over uninfected controls and were calculated by the ΔΔCT method using 18S as the reference gene.
For absolute viral nucleoprotein quantification, RNA was extracted from 1 x 10⁷ PFU PR8 using the ISOLATEII RNA kit according to the manufacturer’s instructions (Bioline) and 100 ng reverse-transcribed with the Tetro cDNA synthesis Kit using IAV nucleoprotein specific primers [39 (link)]. Following amplification, the 216 bp cDNA product was gel purified using a Gel Extraction kit (Sigma Aldrich) and total copy number determined based on size and yield of product. A standard curve was generated to determine absolute viral nucleoprotein mRNA copy number among sample mRNA.
All quantitative reverse-transcriptase PCR (qRT-PCR) was performed using SYBR NoROX master mix (Bioline) on a Roche LightCycler480. Forward and reverse qRT-PCR primers are listed in Table 1.

Stifter S.A., Bhattacharyya N., Pillay R., Flórido M., Triccas J.A., Britton W.J, & Feng C.G. (2016). Functional Interplay between Type I and II Interferons Is Essential to Limit Influenza A Virus-Induced Tissue Inflammation. PLoS Pathogens, 12(1), e1005378.

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Quantifying Hematopoietic Progenitor Gene Expression

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On GD 11.5, hematopoietic cells pooled from individual litters of vehicle- or TCDD-exposed fetuses were isolated and sorted directly into Trizol (Life Technologies) using c-Kit and DCF fluorescence to discriminate between hematopoietic progenitor cells with long- and short-term self-renewal potential, respectively. RNA was purified, quantified and processed as previously described (Ahrenhoerster et al. 2014 (link)). RNA was subjected to reverse transcription using a Tetro cDNA Synthesis kit (Bioline) with both anchored-oligo(dT) 18 priming and random hexamer priming options and was stored at –20°C until the day of the assay. Primers were selected using Universal ProbeLibrary v.2.5 for Mouse (Roche) and checked for specificity using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Gene names, accession numbers, and primer sequences are provided in Table S1. cDNA was used as a template in a 20-μL reaction consisting of 10 μmol of forward and reverse primers and 10 μL SensiFAST SYBR No-ROX (Bioline). Relative expression change was determined using the 2^–ΔΔC_T method with standardization to housekeeping genes. Samples were run in triplicate wells with at least two independent litters per treatment analyzed. Cycling conditions were 95°C for 2 min, followed by 45 cycles at 95°C for 5 sec, 60°C for 10 sec, and 72°C for 10 sec.

Laiosa M.D., Tate E.R., Ahrenhoerster L.S., Chen Y, & Wang D. (2015). Effects of Developmental Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Long-term Self-renewal of Murine Hematopoietic Stem Cells. Environmental Health Perspectives, 124(7), 957-965.

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Quantifying Osmolyte Transporter Expression

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RNA was extracted from NHEK cell pellets from each donor using the RNAeasy mini kit (Qiagen) according to manufacturer's protocol. RNA was converted to cDNA using the Tetro cDNA synthesis kit (Bioline) according to manufacturer's protocol. For each sample, qRT–PCR was performed in triplicate using Taqman probes for the osmolyte transporters BGT‐1, HMIT, SMIT, or TAUT (see Appendix S1), and reactions were performed on the StepOnePlus RT–PCR machine. Relative percentage mRNA expression was calculated for each gene of interest against the housekeeping gene (PPIA) using the ΔCT method.

Foster A.R., El Chami C., O'Neill C.A, & Watson R.E. (2019). Osmolyte transporter expression is reduced in photoaged human skin: Implications for skin hydration in aging. Aging Cell, 19(1), e13058.

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Real-Time PCR Analysis of Mouse Liver Genes

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Total RNA was isolated from mouse liver by using the TRIzol^® (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. Three micrograms of RNA were reverse-transcribed by using Tetro cDNA Synthesis kit (BIOLINE, Taunton, Massachusetts). The real-time PCR reactions were performed by using KAPA SYBR^® FAST qPCR Kits (Kapa Biosystems, Wilmington, Massachusetts). Primer sequences are listed in Table 1.Table 1

Primer sequences.

Gene name	Species	Sequence
eGFP	Firefly	F: 5′- CACCATCTTCTTCAAGGACGA-3′
eGFP	Firefly	R: 5′- TGATGCCGTTCTTCTGC-3′
GNMT	Human	F: 5′- ACTGGATGACTCTGGACAA-3′
GNMT	Human	R: 5′- ACTGAGGATGTGGTCGT-3′
GNMT	Mouse	F: 5′- GTTGACGCTGGACAAAGA-3′
GNMT	Mouse	R: 5′- AGCCTGTGCTGAGGATA −3′
F4/80	Mouse	F: 5′- CAAGACTGACAACCAGACG-3′
F4/80	Mouse	R: 5′- ACAGAAGCAGAGATTATGACC-3′
TNF-α	Mouse	F: 5′- GCCTCTTCTCATTCCTGCTTG-3′
TNF-α	Mouse	R: 5′- CTGATGAGAGGGAGGCCATT-3′
IL-6	Mouse	F: 5′- GGACTGATGCTGGTGAC-3′
IL-6	Mouse	R: 5′- CATTTCTTTGTATCTCTGGAAGTT-3′
Collagen I	Mouse	F: 5′- AAGAGGCGAGAGAGGTT-3′
Collagen I	Mouse	R: 5′- CCTTTGGGACCAGCATC-3′
TIMP1	Mouse	F: 5′- CACAAGTCCCAGAACCG-3′
TIMP1	Mouse	R: 5′- GTCCACAAACAGTGAGTGTC-3′
α-SMA	Mouse	F: 5′- ATTCAGGCTGTGCTGTC-3′
α-SMA	Mouse	R: 5′- TCTCACGCTCGGCAGTA-3′
Desmin	Mouse	F: 5′- CAGGCAGCCAATAAGAAC-3′
Desmin	Mouse	R: 5′- GCCATCTCATCCTTTAGGT-3′
TGF-β1	Mouse	F: 5′- CCAAAGACATCTCACACAGTA-3′
TGF-β1	Mouse	R: 5′- GCCACTCAGGCGTATCA-3′
MMP13	Mouse	F: 5′- TGCCATTACCAGTCTCCG-3′
MMP13	Mouse	R: 5′- TGTCATAACCATTCAGAGCCC-3′
β-actin	Mouse	F: 5′- TGCTCGGGACGTTCACAAC-3′
β-actin	Mouse	R: 5′- GAGAATAAAGCAACTGCACAAACAA-3′

Fang C.C., Wu C.F., Liao Y.J., Huang S.F., Chen M, & Chen Y.M. (2018). AAV serotype 8-mediated liver specific GNMT expression delays progression of hepatocellular carcinoma and prevents carbon tetrachloride-induced liver damage. Scientific Reports, 8, 13802.

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Validating RPKM Values via RT-PCR

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Expression analysis to validate RPKM values that were previously calculated^{17 ,26 (link)–28 (link)} was performed using RT-PCR as previously described^{26 (link)} with slight modifications. In brief, S. verreauxi samples were obtained from cultured animals supplied by Institute for Marine and Antarctic Studies, University of Tasmania, Hobart. Lobsters were reared as previously described^{29 (link),30 (link)}. Total RNA was isolated from whole phyllosoma and puerulus individuals using Trizol® Reagent (Invitrogen), according to manufacturer’s instructions, followed by reverse transcriptase reaction containing 1 μg total RNA extracted from each individual, using Tetro cDNA Synthesis Kit (Bioline, Australia) following manufacturer’s instructions. The cDNA was then amplified by PCR using F1 polymerase (Fisher Biotec, Australia) using 1 μL cDNA as a template, 100 nM of forward (F) and reverse (R) specific primers under the following conditions: 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, annealing at 60 °C for 30 s, elongation at 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. Primers were designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) and produced by Sigma–Aldrich. Amplicons were then electrophoresed on a 1.5% agarose gel stained with ethidium bromide and visualized under UV light.

Ventura T., Nguyen C., Fitzgibbon Q.P., Abramov T., Smith G.G, & Elizur A. (2019). Crustacean larval factor shares structural characteristics with the insect-specific follicle cell protein. Scientific Reports, 9, 2847.

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Physcomitrium patens Transcriptome Analysis

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Physcomitrium patens RNA was isolated as described in Vesty et al. (2016 (link)). Physcomitrium patens cDNA was synthesised using the Tetro cDNA synthesis kit (Bioline) with OligodT primers as per manufacturer's instructions. Reverse transcription PCR (RT‐PCR) was performed as described in Vesty et al. (2016 (link)).

Phokas A., Meyberg R., Briones‐Moreno A., Hernandez‐Garcia J., Wadsworth P.T., Vesty E.F., Blazquez M.A., Rensing S.A, & Coates J.C. (2023). DELLA proteins regulate spore germination and reproductive development in Physcomitrium patens. The New Phytologist, 238(2), 654-672.

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Quantitative Gene Expression Analysis

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Shoots of 14‐day‐old seedlings were snap frozen in liquid N. Total RNA was extracted from frozen tissue using Isolate II Plant RNA Kit (Bioline) with on‐column DNase I treatment. cDNA was prepared from 0.5 μg RNA with Tetro cDNA Synthesis Kit (Bioline, Alexandria, NSW, Australia) using oligo‐dT primer. Technical replicates of gene‐specific products were amplified with primers in Table S2 in 10 μl reactions using SensiFAST SYBR no‐ROX kit (Bioline) on a CFX96 Thermocycler (Bio‐Rad). Transcript levels were calculated from C_t values, incorporating PCR efficiencies calculated with LinRegPCR (Ruijter et al., 2009), relative to the geometric mean of two reference genes ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2) and PROTEIN PHOSPHATASE 2A SUBUNIT A3 (PP2AA3) (Vandesompele et al., 2002).

Román Á., Golz J.F., Webb A.A., Graham I.A, & Haydon M.J. (2019). Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis. The Plant Journal, 102(1), 187-198.

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Quantitative RT-PCR Analysis of Liver Genes

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RNA was isolated with QIAzol Lysis Reagent (QIAGEN, Chadstone, VIC Australia, Cat. 79306) according to the manufacturer's instructions. cDNA was synthesized from 2 µg of total RNA with the Tetro cDNA Synthesis Kit (Bioline, Alexandria, NSW, Australia, Cat. 65050). PCR was performed with DNA polymerase (Bioline, Cat. 21040). The following primer pairs were used to specifically amplify alpha fetoprotein (Afp; Fwd: 5′-TCGTATTCCAACAGGAGG-3′, Rev: 5′-AGGCTTTTGCTTCACCAG-3′), albumin (Alb; Fwd: 5′-CTTAAACCGATGGGCGATCTCACT-3′, Rev: 5′-CCCCACTAGCCTCTGGCAAAAT-3′), glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Fwd: 5′-TGTTCCTACCCCCAATGTGT-3′, Rev: 5′-TGTGAGGGAGATRGCTCAGTG-3′), hepatocyte nuclear factor homeobox 1B (Hnf1b; Fwd: 5′-CAGCCAGTCGGTTTTACAGC-3′, Rev: 5′-TCCTCCCGACACTGTGATCT-3′), Hnf4α (Fwd: 5′-CAGCAATGGACAGATGTGTGA-3′, Rev: 5′-TGGTGATGGCTGTGGAGTC-3′) and tyrosine aminotransferase (Tat; Fwd: 5′-GGGTTGTCTGCCATTCCT-3′, Rev: 5′-TTCTGGGAAGTGCTCCATCT-3′). Primers were synthesized by Geneworks (Hindmarsh, SA, Australia). PCR amplicons were separated on agarose gels and visualized using a ChemiDoc XRS (Bio-Rad). All PCR products were verified by sequencing for authenticity.

Passman A.M., Strauss R.P., McSpadden S.B., Finch-Edmondson M.L., Woo K.H., Diepeveen L.A., London R., Callus B.A, & Yeoh G.C. (2015). A modified choline-deficient, ethionine-supplemented diet reduces morbidity and retains a liver progenitor cell response in mice. Disease Models & Mechanisms, 8(12), 1635-1641.

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Quantitative Real-Time PCR for Gene Expression

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A Tetro cDNA synthesis kit (catalogue no. BIO-65043, Bioline, London, UK) was used to synthesise cDNA using random primers, while following the manufacturer’s instructions. Two genes from each condition (YPD-28 °C, YPD-16 °C, and YP + glycerol-16 °C) showing significant transcriptional changes in RNAseq dataset were picked for further validation of the expression by real-time PCR. Real-time PCR was performed in a Roche thermocycler on the cDNA of hybrids and parental strains using the iTaq Universal SYBR Green Supermix (catalogue no. 1725121, BioRad, Deeside, UK). Reverse transcription and real time PCR was done, as described previously [14 (link),15 (link)]. Table S8 shows the primers used for real time.

Hewitt S.K., Duangrattanalert K., Burgis T., Zeef L.A., Naseeb S, & Delneri D. (2020). Plasticity of Mitochondrial DNA Inheritance and Its Impact on Nuclear Gene Transcription in Yeast Hybrids. Microorganisms, 8(4), 494.

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Lymph Node RNA Extraction and qRT-PCR

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LN were collected and submerged in RNAlater (Ambion) for 24 h (4°C) prior to long term storage at -80°C. RNA was purified from lymph nodes using Trisure (Bioline) as per manufacturer’s instructions (Bioline). RNA (2 μg) was reverse transcribed using the Tetro cDNA synthesis kit with random primers according to the manufacturer’s instructions (Bioline). Quantitative reverse transcriptase PCR (qRT-PCR) was performed using SensiFast SYBR Green (Bioline) on a Roche LightCycler480. Data are expressed as fold change over uninfected control lymph nodes. Data were calculated by ΔΔCT method using 18S as the reference gene. Forward and reverse qRT-PCR primers used in this study: 18S Fw GTAACCCGTTGAACCCCATT, Rv CCATCCAATCGGTAGTAGCG, vegfr3 Fw GCGACAGGGTTCTCATAA, Rv CGTTGCCTCATTGTGATTAG, vegfc Fw CAGCCCACCCTCAATACCAG, Rv GCTGCTCCAAACTCCTTCC.

Daniel L., Counoupas C., Bhattacharyya N.D., Triccas J.A., Britton W.J, & Feng C.G. (2023). L-selectin-dependent and -independent homing of naïve lymphocytes through the lung draining lymph node support T cell response to pulmonary Mycobacterium tuberculosis infection. PLOS Pathogens, 19(7), e1011460.

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