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High fidelity cdna synthesis kit

Manufactured by Roche
Sourced in Germany, United States, Switzerland

The High Fidelity cDNA Synthesis Kit is a laboratory product that allows for the efficient conversion of RNA into complementary DNA (cDNA) with high fidelity. The kit includes all the necessary reagents and components required to perform this process.

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19 protocols using high fidelity cdna synthesis kit

1

Characterizing Cellular Response to Infection

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MTT reagent Thiazolyl Blue Tetrazolium Bromide (Sigma Aldrich), All cell culture dishes (Nunc), TRIzol (Ambion), DEPC, Diethyl pyrocarbonate (Ambion), High-Capacity Reverse Transcription Kit (Applied Biosciences, Inc. Foster, CA), DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Scientific), EDTA-free Protease-cocktail inhibitor (Roche Mannheim Germany), Agarose (Invitrogen by Life Technologies), Crystal violet (Sigma-Aldrich), Gelatin (Merck), PFA, Paraformaldehyde (Merck), Mouse monoclonal Anti-N, Nucleocapsid protein of MHV-JHM (monoclonal clone 1-16-1, kindly provided by Julian Leibowitz, Texas A&M, College Station, TX), Anti-CD11b (Abcam; catalog no. ab133357), Anti-Iba1 (Wako, Richmond, VA, USA, Cat no. 019-19741, RRID:AB_839504) antibody, Avidin-biotin immunoperoxidase technique (Vector Laboratories), Refrax mounting medium (Anatech Ltd., MI, USA), Direct-Zol RNA MiniPrep (Zymo Research), Turbo DNA-Free Kit (Life Technology), High Fidelity cDNA Synthesis Kit (Roche), Fast Start Universal Probe Master (Rox) (Roche), Viral-ToxGlo assay (Promega), Prime-direct probe RT-qPCR mix (Takara).
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2

Quantification of Caspase 3 mRNA Levels

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Primers for Caspase 3 F: 5′-GAGGCGGTTGTAGAAGAGTT CGTG-3′ and Caspase 3 R: 5′-TGGGGGAAGAGGCAG GTGCA-3′ were designed by using Primer-BLAST online software from the National Center for Biotechnology (Bethesda, MD, United States) and synthesized by Macrogen (Seoul, South Korea). Total RNAs from all treated and non-treated samples were isolated using TRIzol reagent (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions. High Fidelity cDNA-synthesis kit (Roche, United States) was used to synthsize the cDNA’s. QuantiTect SYBR Green PCR kit (Qiagen, United States) was used for the quantitative polymerase chain reaction (qPCR) to quantify mRNA levels of the genes. The housekeeping gene, 18sRNA (Hs_RRN18S_1_SG QuantiTect Primer Assay) was used for normalization of data. All RT-PCR experiments were done using iCycler RT-PCR system (Bio-Rad, Hercules, CA, United States).
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3

Differential Expression of lncRNAs in Boar Sperm

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The DElncRNA expression profiles of the transcriptome data (Supplementary Table S1) were confirmed using RT-qPCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene [40 (link)]. Primers used for the DElncRNAs (TRINITY_DN1365027_c0_g1_i2, TRINITY_DN1103286_c5_g1_i5 and TRINITY_DN1278737_c0_g1_i1) are shown in Table 1. Total RNA samples, isolated from the spermatozoa of each boar [14 (link)], were reversely transcribed, and cDNA was synthesized in a PCR Thermal Cycler (Labcycler, Sensoquest GmbH, Göttingen, Germany). For quantitative real-time analysis, 100 ng RNA was used as the template, and reactions were performed using the High Fidelity cDNA Synthesis Kit (Roche Diagnostics International, Basel, Switzerland) with random hexamer, according to the manufacturer’s recommendations. Real-time measurements of the amplification products were performed in a Real-Time PCR system (ABI 7900 H T, Applied Biosystems, CA, USA) [41 (link)]. Briefly, the master mix volume comprised 5 µL SYBR Green mix (Maxima SYBR Green/ROX qPCR Master Mix x2, Thermofisher Scientific, USA), 10 µM each forward and reverse primers (2 µL) and 3 µL template cDNA (equivalent amount of 3.75 ng mRNA). The relative quantification of the transcript expression between the freezability groups was measured, using the Real-Time PCR Miner algorithm [42 (link)].
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4

Quantitative Real-Time PCR of Tobacco Gene Expression

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Total RNA was extracted from different tissue samples using Trizol (Invitrogen, CA, USA). Eight-day-old seedlings of tobacco were used for RNA isolation. First strand cDNA was synthesized by High Fidelity cDNA synthesis kit (Roche Diagnostics GmbH, Germany) and oligo dT primer at 50°C for 30 min. Quantitative real-time PCR (qRT-PCR) experiments and calculations were performed using three technical and three biological replicates following the methods described before (Meena et al., 2015 (link)). Briefly, the reaction was performed in 10 μl reaction volume with 225 nM of each of the forward and reverse primers (Supplementary Table 2) and 2X Power SYBR Green PCR master mix (Applied Biosystems, CA, USA) using Vii A 7 Real-Time PCR System (Applied Biosystems, CA, USA). Chickpea Elongation factor 1-α (EF-1α) (GenBank: AJ004960.1) and tobacco actin (GenBank: BAD27408) genes were used as internal controls. Calculations were done using delta-delta Ct method. Paired students t-test was conducted to determine statistical significance of the results.
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5

Quantitative PCR of Neural Lineage Markers

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Total RNA was extracted with High Pure RNA Isolation Kit (Roche), according to the manufacturer’s instructions. RNA was quantified in a NanoDrop 2000c (Thermo Scientific) and used for cDNA synthesis. Reverse transcription was performed with High Fidelity cDNA Synthesis Kit (Roche), using Anchored-oligo(dT)18 Primer (Roche) or with the Super Script III First Strand synthesis system (Invitrogen), using random hexamers (Invitrogen). qPCRs were performed in triplicates using LightCycler 480 SYBR Green I Master Kit (Roche) with the following primers: βIII-tubulin (TUBB3; fwd 5′-GGGCCTTTGGACATCTCTTC-3′ and rev 5′-CCTCCGTGTAGTGACCCTTG-3′), glial fibrillary acidic protein (GFAP; fwd 5′-AGAGAGGTCAAGCCCAGGAG-3′ and rev 5′-GGTCACCCACAACCCCTACT-3′) and ribosomal protein L22 (RPL22; fwd 5′-CACGAAGGAGGAGTGACTGG-3′ and rev 5′-TGTGGCACACCACTGACATT-3′). The reactions were performed with LightCycler 480 Instrument II 96-well block (Roche). Quantification cycle values (Cq’s) and melting curves were determined using LightCycler 480 Software version 1.5 (Roche). All data were analyzed using the 2−ΔΔCt method for relative gene expression analysis47 (link). Changes in gene expression were normalized using the housekeeping gene RPL22 as internal control.
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6

Rat Liver Gene Expression Analysis

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The transcript levels of the seven genes (Usp30, Grb10, Pld1, St6galnac5, Oxct2b, Khk, and Acsl4) were determined in the liver of the three generations of rats from the restricted and control groups. Each generation/diet group was composed of six female fetuses from day 19 of pregnancy. The total RNA was isolated using Trizol (Roche) reagent, following the standard protocol. The quality and quantity of RNA were checked on a Nanodrop spectrophotometer. Reverse transcription was performed using High Fidelity cDNA Synthesis Kit (Roche) following real-time PCR on a Light Cycler 480 Instrument (Roche) using SYBR Green detection format (Roche). Each sample was analyzed in duplicate and the results were normalized using two reference genes—Hprt (hypoxanthine-guanine phosphoribosyltransferase) and Tbp (TATA box binding protein)—while relative transcript quantification was performed using the 2-ΔΔCT method, according to Livak and Schmittgen [12 (link)]. The details of the primer sequences and amplicon lengths are given in Table B in S2 File.
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7

Validation of Microarray Gene Expression

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The differentially expressed genes from microarray results were selected for validation using fluorescent based hydrolysis probes in Light Cycler 480 (Roche Diagnostics Pvt. Ltd, GmbH, Germany). Two independent groups of samples were taken for validating the microarray data. The first group consisted of 30 individuals which were included in microarray study. Validation was not possible for six individuals due to the limitation of samples in that particular aliquot (cDNA prepared for these six samples were insufficient to carry out validation in real time q-PCR). The second group consisted of freshly collected independent set of 24 individuals from Kerala coast. For each sample, total RNA was isolated, checked for purity and integrity. cDNA was synthesized using High fidelity cDNA synthesis Kit (Roche Diagnostics). For all the genes, intron spanning PCR primers were designed using Probe finder software version 1.1 and specific hydrolysis probes from Universal Probe library set for Humans (Roche Diagnostics) were used. Primer sequences used in the study are given in S1 Table. RT-q PCR reactions were performed on 96 multi-well plates in triplicates and ß-actin (ACTB) was used as a housekeeping gene for normalization. Fold change was calculated by dividing average normalized value in HLNRA individuals with average normalized value in NLNRA individuals.
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8

Quantitative RT-PCR Analysis of Gene Expression

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Total RNA (the same RNA samples used for the RNA-seq experiments; 12 samples representing 4 conditions in 3 biological replicates) were treated with DNase, and 1 μg of treated RNA was used as the template for cDNA synthesis using a high-fidelity cDNA synthesis kit (Roche Diagnostics, Switzerland). To determine relative gene expression, real-time quantitative PCR (qPCR) was performed using primers, TaqMan probes (modified with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine), and iTaq supermix with carboxy-X-rhodamine (Bio-Rad AG, Switzerland) in a StepOnePlus real-time PCR system (Applied Biosystems-Life Technologies, Switzerland). Each reaction was run in duplicate. The primers and probes are listed in Table S3. Relative transcript quantities were assessed using the 2−ΔΔCT threshold cycle (CT) method (55 (link)) to determine the level of expression, which was normalized to that of ACT1 as a reference gene.
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9

Quantitative Analysis of Tumor Immune Infiltrates

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Ribonucleic acid (RNA) was extracted from tumor biopsy specimens using Trizol reagent (Invitrogen Inc.) according to manufacturer’s instruction. The acquired RNA samples were then used to make cDNA using High Fidelity cDNA Synthesis Kit (Roche Applied Science), followed by qRT-PCR using primer/probe sets specific for CD8 and FoxP3. β-actin was used as the internal control. Primer sequences for β-actin included: 5′-ggatgcagaaggagatcactg- 3′ and 5′ –cgatccacacggagtacttg- 3′, probe sequence for β-actin: 5′ –ccctggcacccagcacaatg- 3′. The primer sequences for CD8 included: 5′ –ccctgagcaactccatcatgt- 3′ and 5′ –gtgggcttcgctggca- 3′, the probe sequence for CD8: 5′ –tcagccacttcgtgccggtcttc- 3′. The primer sequences for FoxP3 included: 5′ – ggcactcctccaggacag- 3′ and 5′ –gctgatcatggctgggctct- 3′, the probe sequence for FoxP3: 5′ –atttcatgcaccagctctcaacggtgg- 3′. The qRT-PCR was run on an ABI-7300 qPCR machine (Applied Biosystem).
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10

RNA Isolation from P. gingivalis

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For RNA isolation, the cells were grown to OD600 of 0.8∼1.0 and collected by centrifugation at 10,000 g for 10 min at 4 °C. Total RNA was isolated from P. gingivalis culture using the SV Total RNA Isolation kit (Promega, Madison, WI). Complementary DNA (cDNA) was synthesized by using the High Fidelity cDNA synthesis kit according to manufacturer’s protocol (Roche, Indianapolis, IN).
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