Résumé
Objective·To compare the quality of RNA extracted from fresh and formalin-fixed paraffin-embedded (FFPE) brain tissues and to explore the long non-coding RNA (IncRNA) expression level.Methods·FFPE samples stored under various conditions and paired frozen brain tissues were collected and total RNA qualities were then detected.Amplification efficiency (AE) and expression stability of each RNA marker were calculated and analyzed based on real-time quantitative PCR.After selecting reference biomarkers,normalized △ Ct values of candidate makers within different amplicon size were measured to assess the possibility of lncRNA quantification in FFPE tissues.Results·The purity of RNA extracted from FFPE was relatively high,but the RNA integrity was lower than fresh samples.All biomarkers were successfully amplified and amplification efficiencies of long-chain RNA markers were correlated with amplicon sizes,sample treatment and preservation conditions,namely temperature and storage time.5S,miR-9 and miR 125b achieved optimal AE and showed quite stable expression in all specimens,therefore they were chosen as control markers.Compared with fresh samples,the △ Ct values of only 2 lncRNA (HAR1F and MALAT1-L,whose amplicon size were both higher than 200 bp,respectively) increased in the FFPE samples kept in 4 ℃,while in FFPE tissues kept in room temperature,increments of the △ Ct values were significant for most target genes except for short amplicon markers (＜60 bp),which showed consistently stable expression in all brain specimens.Conclusion·RNA integrity is affected by sample treatment and preservation conditions,but IncRNA expression levels in FFPE tissues can be accurately quantificated by using optimal amplicon sizes and considerable reference markers.
Résumé
Objective: To compare the quality of RNA extracted from fresh and formalin-fixed paraffin-embedded (FFPE) brain tissues and to explore the long non-coding RNA (lncRNA) expression level. Methods: FFPE samples stored under various conditions and paired frozen brain tissues were collected and total RNA qualities were then detected. Amplification efficiency (AE) and expression stability of each RNA marker were calculated and analyzed based on real-time quantitative PCR. After selecting reference biomarkers, normalized △ Ct values of candidate makers within different amplicon size were measured to assess the possibility of lncRNA quantification in FFPE tissues. Results: The purity of RNA extracted from FFPE was relatively high, but the RNA integrity was lower than fresh samples. All biomarkers were successfully amplified and amplification efficiencies of long-chain RNA markers were correlated with amplicon sizes, sample treatment and preservation conditions, namely temperature and storage time. 5S, miR-9 and miR-125b achieved optimal AE and showed quite stable expression in all specimens, therefore they were chosen as control markers. Compared with fresh samples, the △ Ct values of only 2 lncRNA (HAR1F and MALAT1-L, whose amplicon size were both higher than 200 bp, respectively) increased in the FFPE samples kept in 4 ℃, while in FFPE tissues kept in room temperature, increments of the △ Ct values were significant for most target genes except for short amplicon markers (<60 bp), which showed consistently stable expression in all brain specimens. Conclusion: RNA integrity is affected by sample treatment and preservation conditions, but lncRNA expression levels in FFPE tissues can be accurately quantificated by using optimal amplicon sizes and considerable reference markers.
Résumé
The purpose of this investigation was to study the spatial changes of the maxillofacial complex following maxillary protraction transmitted to the center of resistance of a dry juvenile human skull by a modified maxillary protraction appliance. Four dry juvenile human skulls (without mandible) with well aligned upper deciduous dentition and early mixed dentition were used as experimental samples. A modified protraction headgear was fabricated from a Delare's facemask, and following an alginate impression, an orthodontic resin maxillary splint was made for each dry skull. Protraction force level was maintained at approximately 1000gm per side for 6 hours. Cephalometric radiographs were taken pre- and post-protraction, and nine reference markers with 1.5 mm length of .017 x .025 TMA wire were placed on the right side of the skull for an accurate superimposition of serial cephalometric radiographs. The present investigation demonstrated that vertical changes associated with an anterior displacement of the maxillary complex was observed, and the most prominent effect of protraction headgear was a counterclockwise rotation of the maxilla, that is, a forward and downward tipping around the palatomaxillary region.