Are increased computational costs associated with "on the fly" ligand flexibility in docking justified or is it enough to prepare a pool of low energy conformers and reduce a rather complex docking procedure to "simple" accommodating of prepared conformers (without further modifying their geometry) into the protein binding site (with side-chains being either rigid or flexible)?
The pSM2 library version 1.3 comprising 15,148 constructs targetting 9,392 human cancer associated genes was amplified and each 96 well plate used to prepare a pool of plasmid DNA; each of the 100 pools contained between 150 to 200 different shRNAmir constructs with each gene being represented by 1 to 3 shRNAmirs.
To test this, we prepared a pool of normalized DNA from the same five individuals previously analyzed separately (pool size of 10 alleles).
Twenty randomly chosen serum samples with known levels of survivin, RF and aCCP were used to prepare a survivin-positive pool (pool A), a RF-positive pool (pool B), a RF- and aCCP-positive pool (pool C) and a negative pool (pool D) (Table 2).
To prepare a cDNA pool from each RNA sample, total RNA (5 μg) was reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI).
Normal tissue was obtained from benign breast tissue outside the tumour from five patients to prepare a reference pool for cDNA microarray analysis.
To prepare a cDNA pool from each RNA sample, total RNA (5 μg) was reverse transcribed using MMLV reverse transcriptase (Promega, Madison, WI), and the resulting samples were diluted 40 times by volume with nuclease-free water.
In an attempt to overcome or minimize such variability in the normal samples we have prepared a RNA pool containing equal amounts of total RNAs extracted from the normal cervical biopsies and we hybridized it on the miRNA arrays as described for the other samples, and performed Taqman miRNA assay.
This difference also made it easy to prepare a saturation mutant gene pool using short oligonucleotides as mentioned in the section below.
Fifty individuals of C. sinicus from the same population (the Yellow Sea) were pooled to prepare a DNA template for mitogenome sequencing.
For relative quantification of gene expression in native cell lines, we prepared an equimolar pool of RNAs from eight normal tissues of different embryonic origin (adrenal gland, bladder, breast, brain, colon, lung, placenta and prostate) (FirstChoice® Human Normal Tissue Total RNA, Ambion) to be employed as reference sample.
