The quality of subtraction was controlled as recommended by the PCR-Select protocol provided by Clontech.
The following steps represent how the Cy5 to Cy3 ratios were calculated including measures to ensure quality control: Subtraction of local background fluorescence from the fluorescence intensity of each of the Cy3 and Cy5 spots.
The quality of the subtraction was assessed by the sequencing of 384 clones (see below).
We took the genes that showed RPKM values of 460.98 and 397.18 respectively, in order to verify the sensitivity and quality of the subtraction.
The quality of the subtraction was evaluated by RT-PCR using primers for the Vascular Endothelial Growth Factor (VEGF) (sense: 5'-ATGAACTTTCTGCTGTCTTGGGTG-3' and antisense: 5'-CTCACCGCCTCGGCTTGTCAC-3'), this gene being highly induced in hypoxic condition.
The detection of several genes already known to be expressed during meiosis, such as Stk31 [EMBL: CU655270], Smc3 [EMBL: CU653776], Dmrt7 [EMBL: CU652558], Zp148 [EMBL: CU655301], Mutl1 [EMBL: CU654415], Dazl [EMBL: CU654830], Stathmin [EMBL: CU652110], Msh2 [EMBL: CU654850] and Msh5 [EMBL: CU654104] in the M library increased confidence in the quality of the subtraction procedure.
HQS: High quality sequences, 1-1: Forward subtraction, 2-2: Reversubtractionion, WW: Well watered, WS: Water stressed plants.
Data analysis included exclusion of low-quality spots, background subtraction to obtain the net signal intensity, data normalization, and calculation of the Cy5/Cy3 ratios.
In this paper, we propose a new strategy, to improve image quality by employing Digital subtraction and so called image obtained is Digital subtraction Angiography (DSA).
The quality of the noise subtraction used has recently been called into question.
