High capacity cdna kit

Manufactured by Thermo Fisher Scientific

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The High Capacity cDNA kit is a laboratory product designed for the reverse transcription of RNA to complementary DNA (cDNA). It provides a solution for the efficient conversion of RNA into cDNA, which is a crucial step in various molecular biology and genetic analyses.

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High Capacity Kit (98 citations) High Capacity cDNA Reverse Transcript Kit (26 citations) High Capacity cDNA kit with RNase inhibitor (11 citations) ABI High Capacity cDNA RT Kit (10 citations) High Capacity cDNA Reverse Transcription Kit with random primers (8 citations) High Capacity RNA to cDNA Conversion Kit (4 citations) High Capacity cDNA Reverse Transcription Kit for RNA (4 citations) High Capacity cDNA Reverse Kit (4 citations) CDNA kits (3 citations) High Capacity cDNA Reverse Transcription Kits with random primers (3 citations)

436 protocols using high capacity cdna kit

Quantifying NOS Isoforms in Cerebral Arteries

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Total RNA was obtained from cerebral arteries of control and TBI animals using a Trizol isolation procedure and reverse transcribed into cDNA with the High Capacity cDNA Kit (Applied Biosystems). Quantitative polymerase chain reaction (qPCR) was performed using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems); iNOS‐, eNOS‐, nNOS‐, and GAPDH‐specific primers; and PerfecCta qPCR supermix (Quanta Biosciences), as reported previously.^{42 (link)} Briefly, a total of 6.6 ng of DNAse I‐treated RNA was reverse transcribed into cDNA using the High Capacity cDNA Kit (Applied Biosystems) in a 20 μL reaction. Moreover, qPCR was performed in duplicate for each sample using 1 μL of cDNA as a template for all NOS targets, 1× PerfeCta qPCR supermix (Quanta Biosciences), and 1× Taqman gene expression assays in a 20 μL reaction. qPCR was carried out in an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) using the following conditions: 45°C for 5 minutes and 95°C for 3 minutes followed by 40 cycles of 15 seconds at 95°C and 45 seconds at 60°C. We amplified GAPDH as a normalizing internal control. To calculate the relative index of gene expression, we used the 2^−ΔΔCt method^{43 (link)} using control samples for calibration.

Villalba N., Sonkusare S.K., Longden T.A., Tran T.L., Sackheim A.M., Nelson M.T., Wellman G.C, & Freeman K. (2014). Traumatic Brain Injury Disrupts Cerebrovascular Tone Through Endothelial Inducible Nitric Oxide Synthase Expression and Nitric Oxide Gain of Function. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 3(6), e001474.

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Quantifying Osteoclast Gene Expression

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Total RNAs were extracted from cells using Trizol reagent (Invitrogen), and reverse transcription was performed with High capacity cDNA kit (Thermo Fisher Scientific). qPCR was performed using Quant Studio3 (Applied Biosystems) with the following TaqMan probes: Flrt2 (Mm03809571_m1), Flrt3 (Mm01328142_m1), Unc5b (Mm0050454_m1), DCstamp (Mm 04209236_m1), CtsK (Mm00484039_m1), Nfatc1 (Mm00479445_ m1), ATP6v0d2 (Mm01222963_m1), 18s (Hs99999901_s1).

Shirakawa J., Takegahara N., Kim H., Lee S.H., Sato K., Yamagishi S, & Choi Y. (2019). Flrt2 is involved in fine-tuning of osteoclast multinucleation. BMB Reports, 52(8), 514-519.

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RNA Expression Analysis by qPCR

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500 ng to 1 µg of total RNA was reverse transcribed using the high capacity cDNA kit (ThermoFisher Scientific). The relative quantity of select mRNAs was determined by qPCR: each 25 ul total PCR reaction contained 2 µl of cDNA diluted 10-fold, 0.2 mM of each specific primer (see Supplementary file 5) and qPCR master mix (Biotool) to a 1X final concentration. The reactions were loaded onto a 7300 real-time PCR system (Applied Biosystems).

Ladam F., Stanney W., Donaldson I.J., Yildiz O., Bobola N, & Sagerström C.G. (2018). TALE factors use two distinct functional modes to control an essential zebrafish gene expression program. eLife, 7, e36144.

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RNA Isolation and qPCR Analysis of Hormonal, miRNA, and siRNA Experiments

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RNA from the hormonal treatment, microRNA, and siRNA experiments was isolated using TRIzol (Thermo Fisher Scientific) according to manufacturer’s instructions. Reverse transcription-PCR was performed using the High Capacity cDNA kit (Thermo Fisher Scientific). The qPCR reaction was conducted using TaqMan® Fast Universal PCR Master Mix without AmpErase UNG (Thermo Fisher Scientific). A list of assays used can be found in Supplementary Table 3. For HSD17B1 the following custom primer/probes were used: forward primer 5’-TAT GCG AGA GTC TGG CGG TT-3’, reverse primer; 5’-TGC ACT GGG CCG CAC T-3’ and the probe 5’-CGA TCA GGC TCA AGT GGA CCC CAA-3’. For all experiments Peptidylprolyl Isomerase A (Cyclophilin A), and beta-actin were used as endogenous controls. Data analyses were performed according to the ΔΔCt method, and relative concentrations were calculated against the appropriate control, detailed in the respective result section.

Hilborn E., Stål O., Alexeyenko A, & Jansson A. (2017). The regulation of hydroxysteroid 17β-dehydrogenase type 1 and 2 gene expression in breast cancer cell lines by estradiol, dihydrotestosterone, microRNAs, and genes related to breast cancer. Oncotarget, 8(37), 62183-62194.

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Molecular Regulation of Tissue Fibrosis

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Sphingosine‐1‐phosphate (S1P) was purchased from Enzo Life Science, (USA Cat# BML‐SL140‐000). Sphingosine kinase inhibitor (SK1‐II) was purchased from Tocris (Cat# 2097). LY2109761 was purchased from Sigma Aldrich (Italy, Cat# SML2051). Periodic acid‐Schiff was purchased from Sigma Aldrich (St Louis, USA Cat # 395B‐1KT). TGF‐β was purchase by R&D (USA, Cat #DB100B). Anti‐actin alpha SMA from Sigma Aldrich (Cat # A5228). Anti‐IL33 was purchased from ThermoFisher (Italy Car# AF3626). Anti‐FGF2 was purchased from Santa Cruz Technology (USA Cat# sc‐74412). Diamino‐benzidinic acid system was purchased from Sigma Aldrich (Italy Cat# D3939). OCT medium was purchased from Tissue‐Tek OCT (Pella Inc. Italy, Cat # 27050). High‐capacity cDNA kit and SYBR Green Real‐Time PCR Master Mix were purchased from ThermoFisher Scientific (Monza, Italy Cat # 4368814 and 4385612). CD90 PE mouse/rat and CD326 APC mouse were purchase from a Milteny iBiotec (Bologna, Italy Cat# 130‐094‐528 and #130‐096‐417). TGF‐β1 protein (Cat #ab50036), anti‐wide spectrum Cytokeratin (Cat #ab9377), rabbit anti‐mouse, Vimentin (Cat # ab92547), goat anti‐mouse IgG H&L FITC GtxMu‐003‐D (Cat # ab96885594) and goat anti‐rabbit IgG H&L DyLight (Cat #ab96886) were purchased from AbCAM (Cambridge, UK). Other compounds (e.g., ovalbumin, DAPI, collagenase) were purchased from Sigma Aldrich (Italy).

Riemma M.A., Cerqua I., Romano B., Irollo E., Bertolino A., Camerlingo R., Granato E., Rea G., Scala S., Terlizzi M., Spaziano G., Sorrentino R., D'Agostino B., Roviezzo F, & Cirino G. (2022). Sphingosine‐1‐phosphate/TGF‐β axis drives epithelial mesenchymal transition in asthma‐like disease. British Journal of Pharmacology, 179(8), 1753-1768.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was transcribed with a High-Capacity cDNA kit (Thermo Fisher Scientific). Quantitative RT-PCR was performed using Fast SYBR™ Green Master Mix on a QuantStudio™5 Real-Time PCR System, in a 384-well format (Thermo Fisher Scientific). The geometric mean of 3 reference genes GAPDH, TBP, and 36B4 was used for normalization. Relative expression of target genes was determined. The specific primers are listed in Additional file 1: Table S3.

Liu H.Y., Giraud A., Seignez C., Ahl D., Guo F., Sedin J., Walden T., Oh J.H., van Pijkeren J.P., Holm L., Roos S., Bertilsson S, & Phillipson M. (2021). Distinct B cell subsets in Peyer’s patches convey probiotic effects by Limosilactobacillus reuteri. Microbiome, 9, 198.

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Quantifying Gene Expression in Human Islets

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RNA was reverse transcribed using M-MLV reverse transcriptase, RNAse H minus (Promega). Quantitative PCR was performed using iQ SYBR Green mix and samples were amplified using the CFX Connect Real-time system (Bio-Rad). Islets of human control and T2D patients were homogenized by vortexing in 700 uL Qiazol lysis buffer and the RNA extracted using the miRNeasy kit (Qiagen) with DNase treatment. 100ng total RNA was used for reverse transcription using the High Capacity cDNA kit with RNAse inhibitor (ThermoFisher). For qPCR, PowerUP SYBR Green Master Mix (Applied Biosystems) was used with assay-specific primers (Supplemental Table 1).

Motterle A., Gattesco S., Peyot M.L., Esguerra J.L., Gomez-Ruiz A., Laybutt D.R., Gilon P., Burdet F., Ibberson M., Eliasson L., Prentki M, & Regazzi R. (2017). Identification of islet-enriched long non-coding RNAs contributing to β-cell failure in type 2 diabetes. Molecular Metabolism, 6(11), 1407-1418.

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Quantitative Gene Expression Analysis

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Reverse transcription PCR was performed using the High Capacity cDNA kit (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. Quantitative PCR (qPCR) was done using TaqMan Gene Expression Assays, on a StepOne Plus Real-Time PCR system (Life Technologies, Carlsbad, CA, USA). Primer-probe pairs used for WT1 (Hs00240913_m1); NAB2 (Hs00195573_m1); IRF8 (Hs00175238_m1); GAPDH (Hs99999905_m1); VDR (Hs 01045843_m1); CNND1 (Hs 00277039_m1); and QPRT (Hs 00204757_m1) were purchased from Applied Biosystems (Foster City, CA, USA) as assays-on-demand. Data were collected and analyzed using the Applied Biosystems StepOne^TMReal-Time PCR Software v2.0. The relative quantification in gene expression was determined using the ΔΔCt method [45 (link)].

Nilsson H.J., Montano G., Ullmark T., Lennartsson A., Drott K., Järvstråt L., Nilsson B., Vidovic K, & Gullberg U. (2017). The transcriptional coregulator NAB2 is a target gene for the Wilms' tumor gene 1 protein (WT1) in leukemic cells. Oncotarget, 8(50), 87136-87150.

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Quantifying Butyrate Transporter Expression

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Proximal colon andpartial hypothalamic brain region (coordinates were −1.6 to −2.3 mm caudal to the Bregma, ±1 mm to the midline, and −7 to −8 mm from the brain surface) were harvested from 14 week-old WKY and SHR. Total RNA was isolated using Trizol, and cDNA was synthesized using Trizol and High Capacity cDNA kit (Thermo Fisher Scientific, Waltham, MA). Real-time PCR was performed and data analyzed as described.^{60 (link)} Primers used for butyrate transporters and SCFA-sensing receptors are listed in Table 1.

Yang T., Magee K.L., Colon-Perez L.M., Larkin R., Liao Y.S., Balazic E., Cowart J.R., Arocha R., Redler T., Febo M., Vickroy T., Martyniuk C.J., Reznikov L.R, & Zubcevic J. (2019). Impaired butyrate absorption in the proximal colon, low serum butyrate and diminished central effects of butyrate on blood pressure in spontaneously hypertensive rats. Acta physiologica (Oxford, England), 226(2), e13256.

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Real-time qPCR Gene Expression Analysis

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To quantify gene expression by real-time quantitative polymerase chain reaction (qRT-PCR), RNA was isolated from cell pellets with the RNeasy Mini Kit (Qiagen, Germantown, MD) and genomic DNA contamination was removed using DNA-free DNA Removal kit (ThermoFisher Scientific). Quality of the extracted RNA was assessed by 1% agarose gel electrophoresis and from the A_260nm/A_280nm absorbance ratio (Nanodrop 1000, ThermoFisher Scientific). Next, cDNA was synthesized using the High Capacity cDNA Kit (ThermoFisher Scientific) with either random (provided by manufacturer) or specific primers (see Primers and probes section below). Finally, processed and unprocessed RNA expression levels were assessed using the ΔΔC_q method^{57 (link)}. The primer/probe sets were designed, and their compatibility validated in silico. A serial dilution approach was used to validate the multiplex reaction. All qPCR reactions were carried out on either a StepOnePlus or QuantStudio 5 thermal cycler (ThermoFisher Scientific). All experiments were run in triplicate and the gene expression levels normalized to the B2M results.

Falabella M., Kolesar J.E., Wallace C., de Jesus D., Sun L., Taguchi Y.V., Wang C., Wang T., Xiang I.M., Alder J.K., Maheshan R., Horne W., Turek-Herman J., Pagano P.J., St. Croix C.M., Sondheimer N., Yatsunyk L.A., Johnson F.B, & Kaufman B.A. (2019). G-quadruplex dynamics contribute to regulation of mitochondrial gene expression. Scientific Reports, 9, 5605.

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