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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 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.
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.
Total RNA of the holobiont was extracted from both 'Driver' and 'Tester' samples with phenol∶chloroform∶isoamyl alcohol (25∶24∶1) and isolated by overnight precipitation at −20°C using 3 M sodium acetate and 20 µg µL−1 glycogen.
The tester samples were then mixed without denaturation in presence of the fresh denatured driver cDNAs and allowed to anneal overnight.
Poly(A+) mRNA from aMPV-infected and control cells were also used conversely as driver and tester samples, respectively (Reverse SSH library).
The DNA reassociation results in the quadratic magnification of expression ratios for the up- and down-regulated genes in control and tester samples.
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