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Only the molecules of the tester sample, which were ligated to the two different adaptors, could be amplified exponentially.
The first-strand and double-strand cDNAs were then synthesized and the synthesized double-strand cDNA of the tester sample was digested with restriction enzyme Rsa I.
The tester sample was the cDNA population from the branchial filaments (BR) and the driver sample was the cDNA population from the body wall tissue (BW).
In the present study, the suppression subtractive hybridization (SSH) method was used to identify differentially expressed genes present in the tester sample and absent or present at lower levels in the driver.
In the TR-BW library, the tester sample was the cDNA population from the trophosome (TR) and the driver sample was the cDNA population from the body wall tissue (BW).
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The companies also provide the tester samples.
The tester samples were then mixed without denaturation in presence of the fresh denatured driver cDNAs and allowed to anneal overnight.
In all studies except the one with T3-treated mice, RNA samples originating from livers of individual animals were labeled and each tester sample was hybridized against a control sample.
The presence of many non-differentially expressed genes in an SSH PCR library may not result from experimental error but maybe due to the absence of significantly differentially expressed genes between the chosen driver and tester samples.
In the first hybridization, an excess of driver was added to each tester samples, leading to the enrichment of differently expressed sequences.
In the T3-study, a pool of tester samples (from 6 different T3-treated animals) was hybridized against a pool of control samples due to limited availability of RNA.
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CEO of Professional Science Editing for Scientists @ prosciediting.com