March 15, 2011 (Vol. 31, No. 6)
Michael Daniels, Ph.D.
triplePrep Kit Allows Isolation and Purification of Three Analytes from a Single Source
When it comes to isolating and purifying DNA, RNA, and protein analytes, there are considerable benefits to be gained in getting all three from a single, undivided sample. These benefits include data consistency, sample preservation, and time savings. The ability to build an accurate picture of the relative levels of nucleic acids and proteins in individual tissue and cell samples brings an extra dimension to quantifying expression levels.
GE Healthcare Life Sciences’ illustra™ triplePrep kit can perform all three purifications from a single starting sample, as part of the same protocol, which saves a significant amount of time. In addition, there are potential benefits in how much information you can get from your sample and how best to utilize the available sample material.
The triplePrep kit uses no more sample than would be required to purify a single analyte by conventional methods, so the immediate benefit in reducing the amount of sample required is clear. If samples cannot be replaced easily or are in short supply, then isolating all three analytes in one process is a valuable alternative. Biobanks and sample libraries can be preserved, and hard-to-replace samples are utilized more effectively.
Few samples can be said to be truly homogeneous, even two separate cell culture plates could have different expression profiles. When samples are more complex, or where a more precise understanding of expression profiles is required, the ability to isolate and purify DNA, RNA, and protein from exactly the same starting material is particularly valuable.
Tissue samples, in particular, may contain multiple cell types, and the proportions of each cell type can vary significantly across the sample, making a reliable comparison difficult. One starting sample for all three analytes enables consistent correlation of purified DNA, RNA, and protein.
In this article, triplePrep is compared with traditional, single-analyte, purification kits. Under these experimental conditions, the results indicate no adverse effect on either yield or purity, and improved yields in some instances.
The optimized buffer systems and purification columns developed for the triplePrep kit mean that from the user’s perspective the purification process remains simple, and will be familiar to users of existing spin column type purification kits (Figure 1). Tissue samples are first disrupted and homogenized to enable effective lysis, while cell culture samples can be mixed directly with the optimized lysis buffer provided with the triplePrep kit. Conveniently, the lysed sample can be stored at -80°C for up to six months, if required for processing at a later date.
DNA and RNA Isolation
DNA is the first analyte to be captured, by passing the prepared lysate through a DNA-binding column using a simple spin procedure. The flow-through from the DNA binding column is retained, and RNA is captured next by transferring the flow-through to an RNA binding column, with a further spin procedure.
Once again, the flow-through from the RNA binding column is retained as this contains the protein fraction, but the protocol recommends that the RNA fraction is further purified first to avoid degradation and maximize yield. RNA purification utilizes the DNase 1 supplied with the triplePrep kit and an initial treatment to remove any DNA that may have carried through from the first column is conducted with the RNA still bound to RNA binding column. An incubation of 10 minutes is sufficient to remove any remaining DNA, following which purification is achieved by washing the column with the supplied wash buffer and then eluting into a fresh RNase-free microcentrifuge tube.
Both wash and spin steps require only a single brief spin to pass the respective buffers through the column. DNA purification is achieved using a similar approach, with two wash steps followed by a single elution. Time taken for DNA and RNA purification is no more than 10 minutes each, and experienced users can run DNA and RNA purification concurrently, further reducing the time required. If only DNA and RNA are required then the protocol can be terminated at this point.
The flow-through retained from the RNA binding column now contains just the protein fraction. Purification of this is based on a straightforward precipitation protocol, the flow-through first being mixed with a supplied precipitation reagent and then spun at 16,000 g to pellet the precipitated proteins. The pellet is washed with pure water before resuspension. The triplePrep kit protocol recommends resuspending in a 2D-DIGE buffer for optimal results, although 7M urea can also be used if required.
Yields are expected to vary depending on the sample type but the key question for researchers with precious samples, who need to maximize yield is how the three-in-one approach compares to traditional methods. The triplePrep kit was compared to established kits and protocols for the purification of DNA, RNA, and protein as individual analytes, with the finding that not only does triplePrep equal these methods in yield but it exceeds them in many cases.
Purity and Reliability
The ease of purification, and the yields expected from different sample and tissue types varies. Those that are more metabolically active contain higher amounts of RNA and protein, while those that are more fibrous in nature are more difficult to disrupt effectively and so tend to have lower overall yields for all three analytes. Any purification method should maintain the overall quality and purity of the isolated analyte despite these differences in the starting sample. Analysis of results from a wide range of starting materials commonly used in research applications shows that the triplePrep kit returns not just high yields but also consistent purity throughout (Table).
The final test of the purified analytes is how they perform in downstream analysis. During testing, the isolated DNA, RNA, and denatured proteins were found to be entirely compatible with end-point PCR, sequencing, real-time PCR, microarray analysis, SDS-PAGE, Western blotting, 2D-DIGE, and LCMS. DNA, RNA, and protein purified using the triplePrep kit were compared with that purified using single-analyte purification methods. In all cases, the data collected from triplePrep samples gave results directly comparable to, or better than, those from traditional approaches. For example, protein analysis was conducted by 2-D DIGE analysis against the recommended 2-D DIGE reference sample (Figure 2).
Isolating and purifying DNA, RNA, and protein from a single sample provides real advantages, not just in the time required to perform the purification itself, but also in reducing the amount of sample that must be used in these processes. This presents a real advantage in situations where samples are in short supply, difficult to replace, or simply unique. For samples whose heterogeneity can lead to questions on how readily RNA, DNA, and protein results can be correlated from separate purification processes, the outcome of using the triplePrep kit is a greater certainty in comparing results and analysis. All of these are possible without any reduction in the yield, quality and performance of the purified analytes.
Michael Daniels, Ph.D. (email@example.com), is global product manager for nucleic acid purification at GE Healthcare Life Sciences.