age: 34 years gender: male cell type: periodontal ligament stem cells
Growth protocol
Periodontitis can impair the osteogenic differentiation of human periodontal mesenchymal stem cells, but the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs (lncRNAs) have been demonstrated to play significant roles under both physiologic and pathological conditions. We performed comprehensive lncRNAs profiling by lncRNA microarray to identify differentially expressed long noncoding RNA expression between Periodontal ligament stem cells from healthy Periodontal tissue and periodontal ligament stem cells from inflammatory periodontal tissue. Our analysis identified 233 lncRNAs and 423 mRNAs that were differently expressed (fold change >2.0, p-value < 0.05) between the two groups of cells. The GO analysis revealed that the significantly down-regulated biological processes included multicellular organismal process, developmental process and multicellular organismal development and the significantly up-regulated biological processes included cellular process, biological regulation and response to stimulus in periodontal ligament stem cells from inflammatory periodontal tissue. The Pathway analysis revealed that the differentially expressed mRNAs may involved in Focal adhesion, ECM-receptor interaction, Bacterial invasion of epithelial cells, Long-term depression, Circadian entrainment and HIF-1 signaling pathway. Two-condition experiment, periodontal ligament stem cells from healthy periodontal tissue (hPDLSCs) vs. periodontal ligament stem cells from inflammatory periodontal tissue (pPDLSCs), Biological replicates: 3 control replicates (hPDLSCs), 3 testing replicates (pPDLSCs). Primary cultures of hPDLSC cultures were obtained from 3 individuals, aged 31-35 years, undergoing routine premolar extractions for orthodontic reasons or third molar extractions. The pPDLSCs were obtain from 3 individuals, aged 27-35 years, diagnosed with stable phase periodontitis with two-thirds alveolar bone loss and more than one pocket (depth > 5 mm). None of these selected subjects had any clinical evidence of recent infection or systemic disease, a history of smoking or histories of maxillofacial surgery, radiotherapy or chemotherapy. Healthy and inflammatory tissues were obtained from the middle 1/3 of teeth roots which washed by sterile phosphate-buffered saline (PBS). These tissues were cutted into small pieces and digested with type 1collagenase (0.66 mg/mL; Sigma, St Louis, MO, USA) for 20 minutes. Then the cell suspensions were filtered and cultured in aminimum essential medium (a-MEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), 0.292 mg/mL of glutamine (Invitrogen, Carlsbad, CA, USA), 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Gibco BRL) at 37℃ in a humidified atmosphere of 5% CO2 and 95% air. After about two weeks, the mixed cells from healthy and inflammatory tissues were digested by trypsin and single cell-derived colony cultures were obtained using the limiting dilution technique. Multiple colony-derived PDLSCs were four passages used in this study.
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the TRIzol® Reagent (Invitrogen life technologies） following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and the RNA integrity was assessed using standard denaturing agarose gel electrophoresis.
Label
33P
Label protocol
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). 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). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
Hybridization protocol
1.5 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Description
Total genomic DNA was spotted on 192 spots for positive control. Nothing was spotted on 40 spot with the aim of negative control. Therefore, a total of 232 spot data were already eliminated from the whole 5584 spot data on the GF202 array.
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Differentially expressed long noncoding RNA expression between periodontal ligament stem cells from healthy periodontal tissue and periodontal ligament stem cells from inflammatory periodontal tissue.
