Normalization of the data of cDNA microarray is an obligatory step during microarray experiments due to the relatively frequent non-specific errors. Generally, normalization of microarray data is based on the null hypothesis and variance model. In the Yang's model (Yang et al., 2001), at least two types of noises are included. The one is additive noise and the other is multiplicative noise. Usually, background is considered as one of additive noise to the signal and the variation between the signal pixels is the representative multiplicative noise. In this study, the relation between the signal (spot intensity minus background intensity) and background was observed and the influence of background on normalization as a representative additive factor was investigated. Although the relation has not been considered as a factor affecting the normalization, it could improve the accuracy of microarray data when the normalization was carried out considering signal/background ratio. The background dependent normalization decreased the number of genes whose expression levels were changed significantly and it could make their distribution more consistent through the whole range of signal intensities. In this study, printing pin dependent normalization was also carried out regarding the printing pin as a representative multiplicative noise. It improved the distribution of spots in the Cy3-Cy5 scatter plot, but its effect was slight. These studies suggest that there are some influences of the signals on the local backgrounds and they must be considered for the normalization of cDNA microarray data.
Carbocyanines ; DNA, Complementary/*analysis/genetics ; Gene Expression Profiling/*methods/*standards ; Linear Models ; Oligonucleotide Array Sequence Analysis/*methods/*standards ; Reference Standards ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVEThe quality of microarray data influences the accuracy of comparative genomic analyses to a large extent. To ensure that the results obtained by using an in situ synthesized microarray are accurate, data quality is to be assessed by evaluating the melting temperature (Tm) of probes, probability of false synthesis rates, and fragmentation of labeled targets.

METHODSDNA from the Yersinia pestis vaccine strain EV76 was used for microarray analyses. Microarray results were confirmed by PCR. Statistical and bioinformatics methods were employed to perform microarray data analyses and evaluation.

RESULTSCorrelation coefficients of the three datasets were above 0.95 after two-time stripping and hybridization with a labeled DNA with the size of fragmentation being 200 bp - 2 kb, which showed that the hybridization results were highly reproducible. Correlation coefficients were lower with the values ranging from 0.87 to 0.92 between the datasets generated from hybridization with different sizes of the labeled DNA fragment. For the relationship between Tm and signal intensity, there was a different distribution of Tm in the lowest 300 or 3,000 probes with a range of 70 °C-72 °C and the highest 300 or 3,000 probes with a range of 72 °C-74 °C.

CONCLUSIONThe results of this study suggest that the initial microarray design may affect the accuracy of final analyses and that the probe Tm and the size of the labeled fragment may be the two factors of the greatest importance.

Cluster Analysis ; Comparative Genomic Hybridization ; methods ; standards ; DNA Fragmentation ; DNA, Bacterial ; genetics ; Genome, Bacterial ; Oligonucleotide Array Sequence Analysis ; methods ; standards ; Polymerase Chain Reaction ; Reproducibility of Results ; Yersinia pestis ; genetics

OBJECTIVE: To assess the clinical application of single nucleotide polymorphism microarray (SNP array) in prenatal genetic diagnosis for fetuses with absent nasal bone. METHODS: Seventy four fetuses with absent nasal bone detected by prenatal ultrasound scanning were recruited from Women's Hospital, Zhejiang University School of Medicine during June 2015 and October 2018. The chromosome karyotypes analysis and SNP array were performed. The correlation between absent fetal nasal bone and chromosome copy number variants was analyzed. RESULTS: Among 74 fetuses, 19 were detected to have chromosomal abnormalities, including 16 cases of trisomy-21, 1 case of trisomy-18 and two cases of micro-deletion/duplication. Among 46 cases with isolated absence of nasal bone, 3 had trisomy-21, and 1 had a micro-duplication. Absence of nasal bone in association with nuchal translucency thickening had a higher rate of abnormal karyotypes compared with isolated absence of nasal bone (=32.27,<0.01). CONCLUSIONS Fetuses with absent nasal bone and nuchal translucency thickening are likely to have chromosome abnormalities, and SNP array testing is recommended to exclude the chromosome abnormalities.
Chromosome Aberrations ; Female ; Fetus ; Humans ; Nasal Bone ; abnormalities ; Oligonucleotide Array Sequence Analysis ; standards ; Polymorphism, Single Nucleotide ; genetics ; Pregnancy ; Pregnancy Trimester, First ; Prenatal Diagnosis ; methods