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Differential expression of selected genes found to be significantly different in the microarray analyses was confirmed by real-time RT-PCR.
RNA for microarray analyses was extracted from three independent replicate cultures of U937T_pUHD10S cells using the RNeasy Plus mini kit (Qiagen) according to the manufacturer's instructions.
An aliquot of each of the same mRNA extractions used for microarray analyses was subsequently treated with DNA-free DNase (Ambion, Austin, TX, USA) according to the manufacturer's recommendations until RNA samples were totally DNA-free when checked by PCR using gapA (a housekeeping gene) primers.
The microarray analyses was primarily based on our previous original Affymetrix microarray data.
The expression of a selection of nine genes from the microarray analyses was validated using reverse-transcription quantitative PCR (RT-qPCR).
The Pearson correlation (r) between the three microarray analyses was high, and varied between 0.91 and 0.93, and was independent of the slide series.
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In both experiments, full genome microarray analyses were carried out on RNA extracted from whole animals recovered from the flight.
Subsequently, microarray analyses were validated by RT-PCR and qRT-PCR.
The microarray analyses were performed as described previously (Hanada et al. [2013]) with the exception that two biological replicates were used.
Microarray analyses were performed after one week of Fe deficiency and excess treatment and at this point, plants correspond to a new transcriptomic/metabolic steady state.
Cytogenetic analyses like G-banded kandotype and chromosomal microarray analyses are often performed to further investigate the chromosome status of a miscarried fetus.
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