Maxima first strand synthesis kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, New Zealand

The Maxima First Strand Synthesis Kit is a tool for the synthesis of first-strand cDNA from total RNA or poly(A)+ RNA. It contains reagents necessary for the reverse transcription reaction.

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13 protocols using maxima first strand synthesis kit

Quantitative Gene Expression Analysis

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Total RNA was extracted from the tumors using the Qiagen RNEASY Mini Kit (Qiagen, Germany). Then, 1 μg of the RNA was converted to cDNA, using the First Strand Maxima Synthesis Kit (Thermo Scientific, USA) on a thermal cycler (Labnet, USA). Next, the real-time polymerase chain reaction was conducted using the Power SybrGreen Kit (Invitrogen, USA) on the iCyler IQ5 (Bio-Rad, USA). The sequence of the primers used is detailed in Table 1. The analysis was done using the iQ5 icycler software, using three housekeeping genes: HPRT, ACTB, and GAPDH.

Abu N., Mohamed N.E., Yeap S.K., Lim K.L., Akhtar M.N., Zulfadli A.J., Kee B.B., Abdullah M.P., Omar A.R, & Alitheen N.B. (2015). In vivo antitumor and antimetastatic effects of flavokawain B in 4T1 breast cancer cell-challenged mice. Drug Design, Development and Therapy, 9, 1401-1417.

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RNA Extraction and RT-qPCR Analysis of Embryonic Samples

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RNA was isolated from 25–30 embryos of each condition from the same experiment using Trizol according to manufacturer’s protocol. 500 ng isolated RNA was reverse transcribed via First Strand Maxima Synthesis kit (ThermoFisher). RT-qPCR was performed using SYBR Green Mix (Bioline) with previously tested primers on a BioRAD CFX Connect. Tests were run in duplicates of at least three independent experiments. Expression was normalized to Elongation factor 1 alpha expression. S1 Table shows the list of studied genes and used primer pairs.

Breuer M., Guglielmi L., Zielonka M., Hemberger V., Kölker S., Okun J.G., Hoffmann G.F., Carl M., Sauer S.W, & Opladen T. (2019). QDPR homologues in Danio rerio regulate melanin synthesis, early gliogenesis, and glutamine homeostasis. PLoS ONE, 14(4), e0215162.

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qPCR and Droplet-based RNA Analysis

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For qPCR analysis, P19 cells were transfected with the appropriate expression constructs for 24 h and total RNA was isolated using the Qiagen RNeasy kit. cDNA synthesis was performed using random primers with the Maxima first strand synthesis kit (Thermo Scientific). Real‐time qPCR was conducted using SYBR green and Thermo Fisher Quant Studio 6 machine. PCR primers are listed Table EV2.
For droplet experiments, RNA from E14 embryonic cortices was isolated using the Qiagen RNeasy kit, quantified using Qubit^® RNA HS Assay Kits (Life Technologies) according to the manufacturer, and snap‐frozen in small aliquots until use.

Esgleas M., Falk S., Forné I., Thiry M., Najas S., Zhang S., Mas‐Sanchez A., Geerlof A., Niessing D., Wang Z., Imhof A, & Götz M. (2020). Trnp1 organizes diverse nuclear membrane‐less compartments in neural stem cells. The EMBO Journal, 39(16), e103373.

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Quantitative Real-Time PCR Analysis in Brassica and Arabidopsis

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One microgram of total RNA from each biological replicate was used to construct cDNA libraries for quantitative real-time PCR (qPCR) using the Maxima First Strand Synthesis Kit (https://www.thermofisher.com/). qPCR was carried out using SsoFast Evagreen Supermix (http://www.bio-rad.com/), following manufacturer’s instructions, but proportionally adjusting to a 10µL reaction volume. Reaction steps were performed as follows: 95°C for 30 seconds, followed by 45 cycles of 95°C for 2 seconds and 60°C for 5 seconds. The ΔΔCt method, using the ubiquitin family protein BnaC08g11930D as a housekeeping gene in B. napus, was used to calculate fold changes between mock and S. sclerotiorum inoculated treatments in both cultivars. For Arabidopsis experiments, EF1ALFA (AT5G60390) was used as a housekeeping gene (Nicot et al., 2005 (link)). Statistical significance was calculated using the Students t-test (P<0.05) and all primer sequences used are presented in Table S9.

Girard I.J., Tong C., Becker M.G., Mao X., Huang J., de Kievit T., Fernando W.G., Liu S, & Belmonte M.F. (2017). RNA sequencing of Brassica napus reveals cellular redox control of Sclerotinia infection. Journal of Experimental Botany, 68(18), 5079-5091.

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Molecular Analysis of Stented PDAs

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Stented PDAs were collected during comprehensive stage 2 surgery, snap frozen in liquid nitrogen and stored at −80°C. Froz en tissues were pulverized in liquid nitrogen, stents removed, and tissues solubilized by mechanical lysis in Trizol at 4°C. Following centrifugation to remove unlysed material, samples were chloroform extracted and aqueous fractions containing RNA purified using RNeasy columns (Qiagen, Inc., Valencia, CA) and the manufacturer’s protocol. RNA was quantified using a Nanodrop 2000 (Thermo Fisher) and equivalent amounts reverse transcribed using the Maxima First Strand Synthesis Kit (Thermo Fisher). Quantitative PCR reactions were performed in duplicate using Thermo Maxima Probe/ROX qPCR Master Mix and Roche Universal Probe/Primer sets. Relative expression was determined using ΔΔC_t analysis with the ribosomal transcript Rpl13a (NM_012423.2) serving as the housekeeping gene (ref below). Genes analyzed for expression were beta-actin (NM_001101.3), alpha-SMA (NM_001141945), transgelin (NM_001001522), smooth muscle calponin (NM_001299), TGFβ1 (NM_000660), Notch 1 (NM_017617.3), Notch 2 (NM_024408), Notch 3 (NM_000435), PDGF-B (NM_002608), Ki67 (NM_002417), PCNA (NM_002592.2), Cyclin D1 (NM_053056.2), p27^Kip1 (NM_004064) and p21^Waf (NM_001220778.1). Primer sequences are listed in Table 4 [12 (link)].

Hibino N., Cismowski M.J., Lilly B.J., McConnell P.I., Shinoka T., Cheatham J.P., Lucchesi P.A., Galantowicz M.E, & Trask A.J. (2015). Potential Molecular Mechanism of Retrograde Aortic Arch Stenosis in the Hybrid Approach to Hypoplastic Left Heart Syndrome. The Annals of thoracic surgery, 100(3), 1013-1020.

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SARS-CoV-2 Viral RNA Quantification

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Vero E6 and pDC were inoculated with SARS-CoV-2 as described above. At the indicated time points, cells were washed thrice with PBS. Vero E6 were further incubated with trypsin 0.25% for 5 min at 37°C to remove cells surface bound particles. Total RNA was extracted using the RNeasy plus mini kit (Qiagen) according to manufacturer’s instruction. cDNAs were generated from 80 ng total RNA by using the Maxima First Strand Synthesis Kit following manufacturer’s instruction (Thermo Fisher Scientific). Amplification products were incubated with 1 Unit of RNAse H for 20 min at 37 °C, followed by 10 min at 72°C for enzyme inactivation, and diluted 10-fold in DNAse/RNAse free water. Real time quantitative PCR was performed using a Power Syber green PCR master Mix (Fisher Thermo Scientific) on a Light Cycler 480 (Roche). The primers used for qPCR were: E_Sarbeco_F1 (5’-ACAGGTACGTTAATAGTTAATAGCGT-3’), E_Sarbeco_R2 (5’-ATATTGCAGCAGTACGCACACA-3’) for viral RNA quantification. The plasmid containing the sequence corresponding to the amplified cDNA was purchased from GenScript (pUC57–2019-nCoV-PC:E; MC_0101078) and serially diluted (294 to 2.94×109 genes copies/μl) to generate standard curves.

Onodi F., Bonnet-Madin L., Meertens L., Karpf L., Poirot J., Zhang S.Y., Picard C., Puel A., Jouanguy E., Zhang Q., Le Goff J., Molina J.M., Delaugerre C., Casanova J.L., Amara A, & Soumelis V. (2021). SARS-CoV-2 induces human plasmacytoid pre-dendritic cell diversification via UNC93B and IRAK4. bioRxiv.

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RNA Isolation and RT-qPCR Protocol

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Total RNA was isolated using the Qiagen RNeasy kit for all qPCR experiments except the FACS-sorted cells. In the case of FACS-sorted cells, PicoPure RNA isolation kit from Thermo Scientific was used to extract RNA. cDNA synthesis was performed using random primers with the Maxima first strand synthesis kit (Thermo Scientific). RT-qPCR was conducted using SYBR Green and a Thermo Fisher Quant Studio 6 machine. The PCR primers used are in Supplemental Table 5.

Ramesh V., Bayam E., Cernilogar F.M., Bonapace I.M., Schulze M., Riemenschneider M.J., Schotta G, & Götz M. (2016). Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration. Genes & Development, 30(19), 2199-2212.

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Actinomycin D Time-Course qPCR

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Cells were treated with 5 ug/ml Actinomycin D (Fisher Scientific). 0, 2, 4, 6, 8, and 12 hr after treatment, RNA was collected in TRIzol (Invitrogen). Reverse transcription was performed with the Maxima first strand synthesis kit (Thermo Scientific). qPCR was then performed with the SensiFAST SYBR Hi-ROX kit (Bioline) on an ABI 7900HT 384-well PCR machine. Each sample was normalized to 18S rRNA and its 0 hr time point.

Freimer J.W., Hu T, & Blelloch R. (2018). Decoupling the impact of microRNAs on translational repression versus RNA degradation in embryonic stem cells. eLife, 7, e38014.

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Quantification of Plant Gene Expression

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Total RNA was extracted from untreated, MgCl₂ mock-treated or Pf Pf0-1(T3S)-infiltrated or Pto DC3000-infiltrated leaves from 4- to 6-week-old Arabidopsis plants via Tri-Reagent [Sigma Aldrich, NZ] and BCP [Sigma Aldrich, NZ] extraction. cDNA was synthesized from RNA using the Maxima First strand synthesis kit following manufacturer’s instructions [Thermo Fisher, NZ]. Quantitative PCR was carried out on a Roche Lightcycler™ LCII using SYBR Green mastermix [Thermo Fisher, NZ]. Primers used for qPCR are listed in Supplementary Table 2.

Jayaraman J., Choi S., Prokchorchik M., Choi D.S., Spiandore A., Rikkerink E.H., Templeton M.D., Segonzac C, & Sohn K.H. (2017). A bacterial acetyltransferase triggers immunity in Arabidopsis thaliana independent of hypersensitive response. Scientific Reports, 7, 3557.

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Quantitative RT-PCR for SARS-CoV-2 Detection

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Vero E6 and pDCs were inoculated with SARS-CoV-2 as described above. At the indicated time points, cells were washed thrice with PBS. Vero E6 were further incubated with trypsin 0.25% for 5 min at 37°C to remove cell surface–bound particles. Total RNA was extracted using the RNeasy plus mini kit (Qiagen) according to the manufacturer’s instructions. cDNAs were generated from 80 ng total RNA by using the Maxima First Strand Synthesis Kit following the manufacturer’s instructions (Thermo Fisher Scientific). Amplification products were incubated with 1 U RNase H for 20 min at 37°C, followed by 10 min at 72°C for enzyme inactivation, and diluted 10-fold in DNase/RNase-free water. Real-time quantitative PCR was performed using a Power Syber Green PCR Master Mix (Thermo Fisher Scientific) on a Light Cycler 480 (Roche). The primers used for qPCR were E_Sarbeco_F1 (5′-ACAGGTACGTTAATAGTTAATAGCGT-3′) and E_Sarbeco_R2 (5′-ATATTGCAGCAGTACGCACACA-3′) for viral RNA quantification. The plasmid containing the sequence corresponding to the amplified cDNA was purchased from GenScript (pUC57-2019-nCoV-PC:E; MC_0101078) and serially diluted (294 to 2.94 × 10⁹ gene copies/µl) to generate standard curves.

Onodi F., Bonnet-Madin L., Meertens L., Karpf L., Poirot J., Zhang S.Y., Picard C., Puel A., Jouanguy E., Zhang Q., Le Goff J., Molina J.M., Delaugerre C., Casanova J.L., Amara A, & Soumelis V. (2021). SARS-CoV-2 induces human plasmacytoid predendritic cell diversification via UNC93B and IRAK4. The Journal of Experimental Medicine, 218(4), e20201387.

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