Micro RNAs（mi RNAs） from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for mi RNA isolation and quantificati...Micro RNAs（mi RNAs） from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for mi RNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of mi RNAs from milk whey. These two methods were modified phenol-based technique（Trizol LS followed by phenol precipitation, the TP method） and combined phenol and column-based approach（Trizol LS followed by cleanup using the mi RNeasy kit, the TM method）. Yield and quality of RNA were rigorously measured using a Nano Drop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous mi RNAs（bta-mi R-141, bta-mi R-146 a, bta-mi R-148 a, bta-mi R-200 c, bta-mi R-362, and bta-mi R-375） and an exogenous spike-in synthetic control mi RNA（cel-mi R-39） were quantified by real-time polymerase chain reaction（PCR） to examine the apparent recovery efficiency of milk whey mi RNAs. Both methods could successfully isolate sufficient small RNA（200 nt） from milk whey, and their yields were quite similar. However, the quantification results show that the total mi RNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey mi RNA due to its consistency and good repeatability in endogenous and spike-in mi RNA recovery. Additionally, quantitative recovery analysis of a spike-in mi RNA may be more accurate to reflect the milk whey mi RNA recovery efficiency than using traditional RNA quality analysis instruments（Nano Drop or Bioanalyzer 2100）.展开更多
基金Project supported by the Zhejiang Provincial Key Science and Technology Innovation Team(No.2011R50025)the National Natural Science Foundation of China(No.31328022)
文摘Micro RNAs（mi RNAs） from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for mi RNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of mi RNAs from milk whey. These two methods were modified phenol-based technique（Trizol LS followed by phenol precipitation, the TP method） and combined phenol and column-based approach（Trizol LS followed by cleanup using the mi RNeasy kit, the TM method）. Yield and quality of RNA were rigorously measured using a Nano Drop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous mi RNAs（bta-mi R-141, bta-mi R-146 a, bta-mi R-148 a, bta-mi R-200 c, bta-mi R-362, and bta-mi R-375） and an exogenous spike-in synthetic control mi RNA（cel-mi R-39） were quantified by real-time polymerase chain reaction（PCR） to examine the apparent recovery efficiency of milk whey mi RNAs. Both methods could successfully isolate sufficient small RNA（200 nt） from milk whey, and their yields were quite similar. However, the quantification results show that the total mi RNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey mi RNA due to its consistency and good repeatability in endogenous and spike-in mi RNA recovery. Additionally, quantitative recovery analysis of a spike-in mi RNA may be more accurate to reflect the milk whey mi RNA recovery efficiency than using traditional RNA quality analysis instruments（Nano Drop or Bioanalyzer 2100）.
