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A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones
Published on: September 16, 2019
Comparison of the MagNA pure LC automated system and the RiboPure-Blood RNA manual method for RNA extraction from
G Garcia-Effron1, S Gamarra, A Crooke
1Laboratorio de Biología Molecular, Servicio de Hematología, Hospital Universitario, Madrid, Spain. guillermo-garcia@ciudad.com.ar
Abstract:
A total of 62 frozen bone marrow specimens conserved in RNA later (Ambion) were processed using two different extraction methods, the MagNA Pure LC system (MAG; Roche) and the manual RiboPure-Blood RNA method (RIBO; Ambion); Beta glucoronidase RNA (GUS) was amplified by LightCycler PCR to evaluate the quality of both extraction procedures. Less than 1000 GUS copies/ml was detected in 26 of 62 specimens (41.94%) processed by MAG and in five of 62 specimens (8.06%) processed by RIBO. Moreover, RNA recovery from the 62 specimens by MAG is, on average, 2.91 cycle threshold-fold higher than RIBO (P = 0.0008). Furthermore, we compared the extraction times and reagent costs of both methods. In conclusion, RNA extraction using MAG is faster to process 32 samples and less expensive than RIBO but it is not sensitive enough to be employed for research purpose in our laboratory.
Insights
The MagNA Pure LC system (MAG) offers faster, cheaper RNA extraction than the RiboPure-Blood RNA method (RIBO), but exhibits lower sensitivity for research applications.
Area of Science:
- Molecular Biology
- Biotechnology
Background:
- Accurate RNA extraction is crucial for molecular research.
- Evaluating RNA extraction methods is essential for optimizing laboratory workflows.
Purpose of the Study:
- To compare the efficiency and cost-effectiveness of two RNA extraction methods: MagNA Pure LC system (MAG) and RiboPure-Blood RNA method (RIBO).
- To assess the sensitivity of both methods for detecting Beta glucoronidase RNA (GUS) in bone marrow specimens.
Main Methods:
- Sixty-two frozen bone marrow specimens were processed using MAG and RIBO extraction methods.
- Beta glucoronidase RNA (GUS) was quantified using LightCycler PCR to evaluate RNA quality and recovery.
- Extraction times and reagent costs were compared between the two methods.
Main Results:
- The MAG method extracted RNA more efficiently, with an average of 2.91 cycle threshold-fold higher recovery than RIBO (P = 0.0008).
- However, the MAG method detected less than 1000 GUS copies/ml in 41.94% of specimens, compared to 8.06% for RIBO.
- MAG processing was faster and less expensive per 32 samples.
Conclusions:
- While the MAG system is cost-effective and faster for RNA extraction, its lower sensitivity makes it unsuitable for research requiring high precision.
- The RiboPure-Blood RNA method, though slower and more expensive, provides superior sensitivity for research purposes.
