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Undivided attention

May 7, 2009

Feature

Uncategorised

In Depth

When it comes to the isolation of genomic DNA, total RNA and total denatured protein – the best yield and quality comes from a single undivided sample

Until recently, researchers in fields such as functional genomics, molecular genetics and biomarker studies had to use three separate kits to isolate DNA, RNA and proteins for use as probes and targets in downstream applications such as PCR, restriction enzyme digestion, RT-PCR, 2-D DIGE and LCMS. However, the use of divided samples could potentially skew results due to heterogeneity between different cell and tissue samples. The demand for good correlation between transcript (gene) expression, protein expression, copy number variation, and SNP detection has resulted in the development of kits such as the illustra triplePrep Kit, which enables the isolation of high quality genomic DNA (gDNA), RNA, and total denatured proteins from a single undivided sample of tissue or cells in less than one hour.

The performance of the illustra triplePrep Kit was tested by using animal tissues from different organs (e.g. liver, kidney, spleen, brain, lung and intestine) or cultured mammalian cells (e.g. HeLa, NIH-3T3, CHO-K1 and HEK-293). Analysis of data relating to yield and purity showed superior performance compared to the DNeasy Blood and Tissue Kit and RNeasy Mini Kit from Qiagen and 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE), the reference method for protein isolation. Comparative analysis was carried out based on yield, compared to amount of sample input, purity, speed, and ease of use when following the manufacturer's recommended protocols. Table 1 shows typical yields obtained from different types of cells and tissues. The following results represent isolates from 10mg rat liver tissue as an example

The yield obtained by each method was comparable, with more than 1000μg

Figure 1: triplePrep workflow

protein isolated from 10mg rat liver, and approximately 950μg by the 2-D DIGE reference method. Data generated from application of DIGE and LCMS demonstrates high purity and quality of the isolated protein. Western blot analysis of the protein samples was used to show presence of ß-actin and GAPDH proteins, providing further evidence of the high quality of the sample isolate. When analysed on LC-MS for peptide profiling, the protein samples showed average representation of 3115 ± 176 peptides. The peptides were then separated by nanoRPC on an Ettan MDLC coupled to a Finnigan LTQ Linear Ion Trap mass spectrometer. The LC-MS data was evaluated using DeCyder MS differential analysis software. The base peak ion chromatography of LC-MS showed a good peptide distribution and signal intensity (Figure 4), signifying high purity and quality.

Comparative analysis with the DNeasy Blood and Tissue Kit, RNeasy Mini Kit and 2-D DIGE showed that the illustra tiplePrep Kit isolated genomic DNA, RNA, and total denatured protein respectively with equal or improved yield, purity and quality.

As biomolecules isolated using the illustra triplePrep Kit are of high quality, they are suitable for use in downstream applications such as PCR, RT-PCR, sequencing, Western blotting, and LCMS. Moreover, the workflow is simple for illustra triplePrep and offers up to 70% time saving compared with individual extractions for the three analytes. In addition, more data can be generated from precious limited sample, and as the workflow is flexible, with multiple stop points in the protocol, it is possible to isolate any two or all three analytes. Direct correlation of data derived from genomic DNA, RNA and protein isolated by the illustra triplePrep Kit can be drawn, as the exact same undivided sample is applied.

Methodology

Using the illustra triplePrep Kit, 10mg tissue was lysed from rat liver with lysis buffer. The tissue was then loaded onto a DNA mini column, and gDNA eluted with elution buffer. Acetone was added to flowthrough containing RNA and protein, and loaded onto an RNA mini column. Following RNA binding to the second column, DNase was used to remove any remaining gDNA contamination. The flowthrough from the RNA column containing only proteins was isolated by precipitation, washed with distilled water and re-suspended in 2-D DIGE buffer. Figure 1 shows the complete triplePrep workflow. Isolated gDNA, RNA and proteins were used in various downstream applications to assess their performance and quality.

Genomic DNA

The illustra triplePrep Kit and DNeasy Kit were used to isolate gDNA from 10mg of rat liver tissue each, and the yield, purity and quality of gDNA obtained by each method were compared. It was shown that the illustra triplePrep Kit achieved 60% higher yield (15.4μg ± 0.60) compared to the DNeasy Blood and Tissue Kit (9.8 μg ± 2.10), using the same amount of input sample (Figure 2). Purity of the gDNA isolated by the illustra triplePrep Kit was determined by A260/A280 optical density ratios (1.90 ± 0.02), indicating good purity. The isolated gDNA was then used to amplify a 1.5kb DNA fragment by PCR, which was sequenced to compare performance in downstream applications.

When 2% of the isolated gDNA was analysed on 0.8% agarose gel, it was demonstrated that the illustra triplePrep Kit gave higher yield and better quality than the DNeasy Kit. In addition, the sequenced PCR fragment generated by the illustra triplePrep Kit showed higher Phred 20 scores (744bp) compared to the DNeasy Blood and Tissue Kit (723bp). The results reflect the suitability and quality of the gDNA isolated by the illustra triplePrep Kit for use in downstream applications such as PCR and sequencing.

Total RNA

Total RNA was isolated from 10mg of rat liver tissue using either the RNeasy Mini kit or the illustra triplePrep Kit, and the results compared for yield, purity and quality. The total RNA samples were used to amplify high, medium and low expressed 18S ribosomal RNA, Cytochrome p450 and c-fos gene respectively by RT-PCR. When assessed by Agilent Bioanalyser, high RNA Integrity Number (RIN) and intact 28S and 18S ribosomal RNA bands indicated high quality RNA (Figure 3). In addition, total RNA isolated with each kit showed similar yield and purity, as determined by A260 and A260/A280 optical density ratios.

Total proteins

According to the illustra triplePrep protocol, proteins were isolated from the second flowthrough, after first isolating gDNA and total RNA. Output from this method was then compared to 2-D DIGE in terms of yield, purity and quality.