Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage (original) (raw)
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A Simple Protein Extraction Method for Proteomic Analysis of Diverse Biological Specimens
Current Proteomics, 2014
The success of a proteomic experiment largely depends on the quality and quantity of the protein extract. Currently, various protocols are available for extraction of proteins from different types of samples; however, further optimization is required for every new sample type. Hence, a common protein extraction protocol is desirable. In the present study, soluble proteins were extracted from six diverse samples using TRIzol without any additional clean-up step and subjected to 2-DE and 2D-DIGE analysis for global protein expression profiling. Image analysis using IMP7 and DeCyder showed good coverage, reproducibility and quality of the gel. MS analysis of 24 spots from all the six samples showed good score and coverage for the identified proteins. Additionally, this method facilitated the concurrent isolation of RNA from the same cell lysates with high integrity and quality, suitable for transcriptomic analysis. Thus, we demonstrate the use of a common protein extraction protocol involving TRIzol reagent for 2-DE, 2D-DIGE and MS analysis using six diverse samples and show its suitability for concomitant transcriptomic studies.
Extraction methods often are chosen to maximize the data that may be obtained such as the simultaneous isolation of messenger RNA (mRNA) 1 and proteins from a limited amount of tissue. Such methods reduce time, costs, and sampling error. More critically, they facilitate more meaningful interpretation and correlation of data. Although several methods for simultaneous RNA-protein extraction are used , it remains diYcult to establish whether divergent results are due to sample preparation and handling or underlying biology. We compared two such established methods routinely used interchangeably for simultaneous extraction of RNA and proteins, Trizol LS reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and guanidinium isothiocyanate (GITC) media , against a third "protein-only" method using two-dimensional (2D) electrophoresis buVer to determine whether similar protein proWles are obtained. Comparisons between Trizol LS and GITC-based RNA extractions have been reported previously [8]; however, a complete lack of information exists regarding the proteins extracted simultaneously using these methods. Both the Trizol LS and GITC methods are modi-Wed versions of the procedure Wrst described by Chomczynski and Sacchi in 1987 [6], and although they use very similar organic solvents and salts, intriguingly diVerent pro-Wles of proteins with very diVerent ranges of molecular sizes are obtained . Trizol LS preferentially extracts lowmolecular weight species, whereas GITC extracts highmolecular weight proteins. These results highlight the importance of using the same analysis protocol for valid comparison and interpretation of results from a sample population. Importantly, the protocols are useful for preferential extraction of low-and high-molecular size proteins, respectively.
Data from Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling
Background: Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription.Methods: GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the “probe level” (a single consensus value) or “bead level” (multiple measurements provided by individual beads).Results: TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbal...
Comparison of phenol-based and alternative RNA isolation methods for gene expression analyses
Journal of the Serbian Chemical Society, 2010
The widespread use of gene expression analyses has been limited by the lack of a critical evaluation of the methods used to extract nucleic acids from human tissues. For evaluating gene expression patterns in whole blood or leukocytes, the method of RNA isolation needs to be considered as a critical variable in the design of the experiment. Quantitative real-time PCR (qPCR) is widely used for the quantification of gene expression in today?s clinical practice. Blood samples as a preferred RNA source for qPCR should be carefully handled and prepared to not inhibit gene expression analyses. The present study was designed to compare the frequently used guanidine thiocyanate-phenol-chloroformbased method (TRI Reagent?) with two alternative RNA isolation methods (6100 PrepStation and QIAamp?) from whole blood or leukocytes for the purpose of gene expression analysis in chronic myeloid leukemia (CML) patients. Based on the results of this study, for the best combination of yield and RNA ex...
The Journal of Pathology, 2009
The development of efficient formaldehyde cross-link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker-driven proteomic investigations. Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of crosslink reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches, attempts at extracting full-length, non-degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat-induced antigen retrieval strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non-degraded, fulllength, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost-effective quantitative option for the validation of potential biomarker panels through a range of clinical samples from existing diagnostic archives, provided that validation of the method is first carried out for the specific proteins under study.
Notes & Tips Improved method for simultaneous isolation of proteins and nucleic acids
Guanidinium thiocyanate–phenol–chloroform extraction (GTPC extraction) is widely used in molecular biology for isolating DNA, RNA, and proteins. Protein isolation by commercially available GTPC solutions is time consuming and the resulting pellets are only incompletely soluble. In this study ethanol–bromo-chloropropane–water was used for precipitation of proteins from the phenol–ethanol phase after GTPC extraction of RNA and DNA. The precipitated proteins can be readily dissolved in 4% SDS for subsequent analysis. This technique allows a fast (30 min) and efficient (with a protein recovery of up to 95%) extraction of proteins for the study of transcriptional and posttranscriptional events from the same sample.
BioTechniques, 2016
Methods for dual extraction of total RNA and protein maximize the biological information obtained from cell or tissue samples while avoiding the need for large sample numbers. Consequently, significant advances have been made in commonly used methodologies, such as the column-based RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada), that permit researchers to study the expression level of a specific gene and its corresponding protein from the same sample (1). These techniques allow for the elution of total RNA with sufficient quality and quantity for microarray analysis and cDNA generation, as well as adequate yields of protein for immunoblots. Moreover, columnbased kits do not require hazardous c he mic a ls, suc h as phe nol or chloroform, or time-consuming cesium chloride (CsCl) gradient centrifugation (1). However, it is not known if protein phosphorylation status is conserved
High-Throughput Sample Preparation from Whole Blood for Gene Expression Analysis
Journal of the Association for Laboratory Automation, 2006
W hole blood is an attractive sample source for nucleic acid-based assays because it is readily available, easily accessible, and rich in genetic information. However, globin mRNA accounts for up to 70% of the mRNA (by mass) in whole blood total RNA, resulting in distortion of the RNA amplification and subsequently causing decreased Present calls, decreased call concordance, and increased signal variation in microarray analysis. Therefore, for gene expression analysis, whole blood is typically fractionated before total RNA isolation to reduce globin mRNA content. We have developed a high-throughput sample preparation technology that streamlines workflows for (1) total RNA isolation from whole blood (MagMAX-96 Blood RNA Isolation Kit), (2) globin mRNA removal using a novel, nonenzymatic technology (GLOBINcleardHuman Kit), and (3) mRNA amplification and labeling for expression analysis (MessageAmp II-96 aRNA Amplification Kit). Globin mRNA removal eliminates the need for prefractionation of whole blood, minimizing the potential for expression profile changes during sample handling. Quantitative RT-PCR showed that this method effectively removed up to 95% of the globin mRNA from the isolated RNA while retaining normal levels of other mRNAs. The streamlined sample preparation enables quick and accurate expression analysis of relatively high numbers of blood samples.
RNA Extraction from Animal and Human's Cancerous Tissues: Does Tissue Matter?
International journal of molecular and cellular medicine, 2015
The reliability of gene expression profiling, based technologies and methods to find transcriptional differences representative of the original samples is influenced by the quality of the extracted RNA. Hence, RNA extraction is the first step to investigate the gene expression and its function. Consequently, the quality of extracted RNA is really significant. Correspondingly, this research was accomplished to optimize the RNA extraction methods and compare the amounts of tissue or quality of tissue. Relatively, the cancerous tissue of human stomach in fresh and frozen conditions and also the mouse fresh tissue were studied. Some factors like the amount of samples, efficacy differences of diverse extraction buffers (TriPure, Trizol) and also the efficacy of b-mercaptoethanol were compared and investigated. The results indicated that the less amount (1-2 mg) compared to other amounts (2-5 mg, 5-15 mg) yielded the best quality and the RNA bands (5S, 18S, 28S) were observed perfectly. R...