ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Polymerase chain reaction (PCR) has become one of the powerful technique since its invention in 1980s. Nevertheless, PCR technique is still frequently impaired by its low specificity, poor sensitivity, false positive results, etc. Recently, nanomaterials including metal nanoparticles, carbon nanomaterials, quantum dots and nano metal oxide have been added into PCR solution to improve both quality and productivity of PCR. The nanoparticles assisted PCR ( NanoPCR) has received considerable attentions due to its unprecedented sensitivity, selectivity and efficiency. In this view, the mainly used nanoparticles in NanoPCR, including gold nanoparticles, quantum dots, carbon nanomaterials, graphene and metallic oxide, was firstly summarized. And then, the possible mechanisms for highly improved sensitivity and selectivity were discussed. Finally, recent applications of NanoPCR were described.