tissue: placenta tissue source: Fresh tissue delivered from preterm premature rupture of membrane gestation: <35GW
Treatment protocol
N/A
Growth protocol
N/A
Extracted molecule
total RNA
Extraction protocol
Total RNA isolated with Trizol. mRNA was purified from total RNA after removal of rRNA with mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre, Omaha, NE).
Label
Cy3
Label protocol
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, Santa Clara, CA) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA with mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre, Omaha, NE). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen, Valencia, CA). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (Agilent Technology, Santa Clara, CA).
Hybridization protocol
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, Santa Clara, CA) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA with mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre, Omaha, NE). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen, Valencia, CA). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (Agilent Technology, Santa Clara, CA).
Scan protocol
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.
Description
PPROM 3
Data processing
Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies, Santa Clara, CA). After normalization of the raw data, lncRNAs and mRNAs that have flags (“All Targets Value”) were chosen for further data analysis. Differentially expressed lncRNAs and mRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Hierarchical Clustering was performed using the Agilent GeneSpring GX software (Version 12.1). Both “GO analysis” and “Pathway analysis” were performed in the standard enrichment computation method.
