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Isolation of Small Noncoding RNAs from Human Serum
Published on: June 19, 2014
A unique method for isolation and solubilization of proteins after extraction of RNA from tumor tissue using trizol
Neah Likhite1, Ujjwala M Warawdekar
1V. G. Vaze College, Mulund, Mumbai, India.
Abstract:
The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at -80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction.
Insights
This study demonstrates a systems approach for simultaneous RNA, DNA, and protein extraction from tumor tissue using Trizol. This method successfully recovers proteins, even from degraded RNA samples, aiding molecular marker analysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Concurrent extraction of RNA, DNA, and protein is crucial for correlating genetic aberrations with proteomic and transcriptomic differences.
- Limited and shared tumor tissue samples necessitate a holistic approach for comprehensive molecular analysis.
- Trizol, a phenol and guanidine isothiocyanate solution, enables simultaneous isolation of multiple biomolecules.
Purpose of the Study:
- To develop and validate a systems approach for simultaneous extraction of RNA, DNA, and protein from tumor tissue.
- To assess the feasibility of protein recovery from Trizol fractions after RNA isolation.
- To correlate molecular marker analysis using salvaged protein from challenging tissue samples.
Main Methods:
- Utilized Trizol for simultaneous RNA, DNA, and protein isolation from non-small cell lung carcinoma (NSCLC) and breast tumor tissues.
- Stored Trizol fractions at -80°C for over 6 months post-RNA extraction.
- Isolated and solubilized proteins using isopropanol precipitation and guanidine hydrochloride/SDS washing, followed by BCA assay and Western blotting.
Main Results:
- Successfully extracted and quantified proteins from 17 tumor and adjacent normal tissue samples, and PBMCs.
- Western blotting confirmed the presence of cytokeratins (CK) and DNA repair proteins.
- Protein analysis salvaged marker analysis in samples with degraded RNA, though nuclear proteins were lost with the DNA fraction.
Conclusions:
- The Trizol-based method provides a viable systems approach for simultaneous biomolecule extraction from limited tumor samples.
- Protein recovery from stored Trizol fractions is feasible and aids in molecular marker analysis, especially when RNA is degraded.
- While effective for cytoplasmic proteins, the method does not retrieve nuclear proteins, which are likely lost with the DNA fraction.
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