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cDNA was used as a template, and quantitative real-time RT-PCR was performed with Bio-rad 170-9780 iQ5 Multicolor Real-Time PCR Detection System (BioRad, USA) according to the manufacturer's protocol.
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Bisulphite-treated DNA was used as a template for MSP and quantitative PCR by ABI 2700 thermocycler (Applied Biosystems, CA, USA) and LightCycler (Roche Diagnostic), respectively.
We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods.
RNAs were 3'-extended with a poly (A) tail, then the first cDNA strand was synthesized and used as template for quantitative real-time RT-PCR with gene specific primers (Table S4) and β-actin was selected as the endogenous reference.
Complementary DNA was transcribed as recommended (Applied Biosystems, Foster City, CA) and used as template for quantitative PCR.
The RNA control was diluted, added to patient sample, and used as template for quantitative real-time RT-PCR as described above.
The cDNA reactions were then diluted to a volume of 50 µl with water and used as a template for quantitative PCR.
First strand cDNA was synthesized from 1 µg of total RNA using QuantiTect Reverse Transcription Kit (Qiagen) and used as a template for quantitative RT-PCR (qRT-PCR).
The extracted RNA was used as template for cDNA generation, which was next applied to a random primed reverse transcriptase reaction (Fermentas, MBI) following the manufacturer's protocol and then used as template for quantitative PCR (Rotor-Gene 6000; Corbett Life Sciences) by means of the SYBR Green I detection system.
The RT reaction mixture was diluted 1/60 and used as cDNA template for quantitative PCR.
cDNAs were generated using purified RNAs (High Capacity cDNA Archive Kit, Life Technologies, Grand Island, NY, USA) and served as the template for quantitative RT-PCR.
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