Total RNA was extracted using the High Pure RNA isolation Kit (Roche) following manufacturer’s guidelines. Total RNA was then quantified by absorption at 260 nm in a Nanodrop spectrophotometer. Samples with 260/280 and 260/230 ratios in the range of 1.8–2 and 2–2.35, respectively, were accepted. Microfluidic RT-Q-PCR was performed in triplicate using the TaqMan Human Stem Cell Pluripotency Array (Applied Biosystems). Regular RT-Q-PCR was performed as previously described [43 (link)] using the ABI PRISM 7900 HT Sequence Detection System. One μg of RNA for each sample was subjected to reverse transcription using the High Capacity cDNA Achieve Kit (Applied Biosystems). For Real Time-Q-PCR 0.5 μg cDNA was amplified using TaqMan based technology (Applied Biosystems). The expression data were normalized taking into account the values of the genes that were expressed in all samples. The normalization formula is as follows: ΔCt(test) = (Ct(gene)-Ct(total average))/SD(Ct(total)).
High capacity cdna achieve kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The High Capacity cDNA Achieve Kit is a laboratory instrument designed for the reverse transcription of RNA into complementary DNA (cDNA). This kit provides the necessary reagents and components to efficiently convert RNA into cDNA, which can then be used for various downstream applications, such as gene expression analysis, cloning, and sequencing.
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Lab products found in correlation
Taqman based technology, by Thermo Fisher Scientific (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Steponeplus software, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna 2, by Macherey-Nagel (1 mentions)
Taqman mgb probe, by Thermo Fisher Scientific (1 mentions)
Taqman pcr kit protocol, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
4 protocols using high capacity cdna achieve kit
1
Comprehensive Stem Cell Transcriptome Analysis
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from the cells using a commercially available kit (NucleoSpin RNAII from Macherey–Nagel, Dueren, Germany), as previously described25 (link). The quantity and purity of the RNA were measured with a Nanodrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France). Total RNA (1 μg) was reverse-transcribed into first-strand cDNA using a High-Capacity cDNA Achieve Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. RT-qPCR was performed using the fluorescent dye SYBR Green method, with SYBR Green PCR Master Mix in 384-well plates and the StepOnePlus system (Applied Biosystems). Amplification curves were analyzed according to the comparative cycle threshold method, using StepOnePlus software (version 2.1, Applied Biosystems by Life Technologies, Paisley, UK). The steady-state mRNA levels for the genes of interest were normalized against those of GAPDH.
3
Quantifying Heme Oxygenase mRNA Levels
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The presence and amount of HO-1 mRNA and HO-2 mRNA were studied by reverse transcription polymerase chain reaction (RT-PCR). RNA obtained from oocytes was transcribed into cDNA with a High Capacity cDNA Achieve Kit (Applied Biosystems, Foster City, CA, USA); the final amount was 100 µl. Based on the knowledge of HO-1 and HO-2 sequences, the specific primers to amplify products were designed (Table S2 ).
Standard TaqMan PCR kit protocol was used (Applied Biosystems, Foster City, CA, USA) for RT-PCR. The reaction ran in a 10 µl reaction mixture, containing 500 nM of gene-specific primers, 200 nM TaqMan MGB probe, 5µl Fast-TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA), and 1 µl cDNA and nuclease-free water. 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) was used for the reaction. Based on the obtained data, the relative amount of mRNA for each isoform was calculated using the 2−ΔΔCT arithmetic equation, according to the Ct method and expressed in comparison to GAPDH as an endogenous control.
Standard TaqMan PCR kit protocol was used (Applied Biosystems, Foster City, CA, USA) for RT-PCR. The reaction ran in a 10 µl reaction mixture, containing 500 nM of gene-specific primers, 200 nM TaqMan MGB probe, 5µl Fast-TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA), and 1 µl cDNA and nuclease-free water. 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) was used for the reaction. Based on the obtained data, the relative amount of mRNA for each isoform was calculated using the 2−ΔΔCT arithmetic equation, according to the Ct method and expressed in comparison to GAPDH as an endogenous control.
4
Transcriptome Analysis of Pseudomonas putida
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Total RNA from stationary phase cultures was extracted as described (García-González et al., 2005) (link) Reverse transcription (RT) of total RNA (2 µg) was carried out using the High-Capacity cDNA Achieve kit (Applied Biosystems) following the manufacturer instructions. Amplification reactions were performed with 50 ng of cDNA as template in a 30-cycle PCR program, using appropriate oligonucleotides detailed in Supplementary Table S2 andIllustra putida KT2440 reference genome (GenBank: NC_002947.4) using Bowtie2 v.2.3.5.1 (Langmead and Salzberg (2012) (link). Mapped data were converted to BAM files using Samtools v1.11 (Li et al., 2009) (link) and reads were visualized with the Integrative Genomics Viewer (IGV) (Robinson et al., 2011) (link). Raw dataset is available at the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) under accession number GEO: GSE173832.
The genome-aligned data were displayed using the Integrated Genomics Viewer (IGV) software.
The genome-aligned data were displayed using the Integrated Genomics Viewer (IGV) software.
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