This blog was written by guest writers Paraj Mandrekar (Technical Services Scientist 3) and Michelle Mandrekar, (Research Scientist 4).
Here are some designer’s notes comparing the Maxwell® RSC Blood DNA and the Maxwell® RSC simplyRNA kit chemistries for nucleic acid extraction.
The Maxwell RSC Blood DNA Kit and Maxwell RSC simplyRNA Blood Kit were both developed from the same non-silica-based purification chemistry and use the same underlying paramagnetic particle. This chemistry is characterized by an extreme binding capacity (the capacity of nucleic acid that can be bound on the particle), leading to both chemistries being capable of isolating large amounts of nucleic acid volumes and then eluting into relatively small volumes (50 µL). It is not unusual with either chemistry to have isolates that exceed 100 ng/µL. Although the chemistries have several similarities, there are some important distinctions between how the two chemistries were designed that influence which kit you choose for your nucleic acid extraction.
Design Notes: Optimized Chemistries for Nucleic Acid Extraction from Blood
Maxwell RSC Blood DNA Kit
The extraction chemistry of the Maxwell RSC Blood DNA Kit was designed for isolating DNA in high concentration from 300 µL of whole blood. In the case of a whole blood sample that sits within the normal range of white blood cell (WBC) count in a volume of whole blood (4–10 million/ml), you could expect to have as much as seven micrograms of DNA available, and this should result in an eluate with a DNA concentration of over 100 ng/µL. This chemistry should then isolate high concentration eluates (100 ng/µL or higher) on a routine basis, if the DNA is available in the sample. Internal testing suggests that this yield may maintain a roughly linear relationship with DNA input volume to around 20 micrograms of yield. This capacity of high bind capacity for DNA, coupled with the 50 µL elution volume combine to result in the high-concentration isolates that characterize this chemistry.
The chemistry was developed around an assumption of isolation from 300 µL of whole blood, combined with 300 µL of the Lysis Buffer, and 30 µL of Proteinase K. The preprocessing consists of 20 minutes of incubation at 56°C. The preprocessing helps to breakdown proteins that might otherwise co-purify on the particle. After incubation, 630 µL of a lysate can be placed directly into the Maxwell RSC Blood DNA cartridge and run on a Maxwell RSC Instrument.
The Maxwell cartridge was optimized around the assumption of adding 630 µL of a lysate into the Maxwell RSC Blood DNA Kit. Smaller volumes of input sample potentially can cause purification issues because one function of the lysis buffer is to dilute the binding buffer (in the cartridge) to the optimum concentration for binding. In our experience, 400 µl of total volume or higher works well in the cartridge.
The chemistry was designed and optimized for DNA isolation, and we know that the underlying chemistry will also bind RNA. However, the preprocessing and binding steps for the Maxwell RSC Blood DNA kit are not optimized to isolate RNA.
Maxwell RSC simplyRNA Blood Kit
This chemistry was designed to purify RNA from white blood cells (WBCs) isolated from 2.5ml whole blood. While the amount of RNA present in a cell can vary based on cell activity, purification of RNA is linear over a wide range of input. A large proportion of RNA in whole blood is hemoglobin RNA (1) from red blood cells (RBCs), which is usually not the RNA of interest in most studies. To reduce the amount of hemoglobin RNA and retain the RNA from the WBCs, the Maxwell RSC simplyRNA Blood Kit uses differential lysis to lyse the RBCs, followed by centrifugation to pellet the intact WBCs.
The WBCs are then treated with 1-thioglycerol, homogenization solution, lysis buffer and proteinase K to lyse the them and inhibit RNases that may be present in the sample to protect the RNA from degradation.
Preprocessing consists of 10 minutes of incubation at room temperature to lyse WBCs, break down the proteins in the sample and release RNA for purification. After incubation all the lysate (approximately 400 µL) is transferred to the Maxwell RSC Cartridge. The binding buffer in the cartridge in combination with the lysate reagents is optimized to maximize RNA binding and reduce DNA binding.
DNase treatment is performed during the Maxwell run which degrades the DNA. The wash solutions in the cartridge were optimized to further reduce the DNA carry through after the DNase treatment. If the DNase is not added, then the RNA purification may not be affected but there will be DNA present in the eluate.
Total Nucleic Acid Extraction Considerations
Both kits are derived from the same underlying chemistry but differ in several important respects. So, if you are trying to isolate both DNA and RNA from a sample, which kit will work best for you?
Both kits will isolate both DNA and RNA, but while the Maxwell RSC Blood DNA kit will isolate DNA efficiently, it may not protect and isolate the RNA in the system. So, if you were trying to get both DNA and RNA, you may want to consider whether you need the DNA or the RNA isolation to be optimized.
Following the protocol for simplyRNA will result in little or no DNA purified but not adding the DNase to the cartridge may allow purification of a significant fraction of the DNA. If a lower DNA yield can be tolerated then simplyRNA without DNase added might be an option for purifying both high quality RNA and DNA.
Nucleic Acid Extraction from Tissue Samples
Promega scientists have developed many applications for many sample types on the Maxwell instrument. While there are many associated application notes than could address a specific sample type, we haven’t tested every sample that could be out there.
We already have a Maxwell RSC simplyRNA Tissue kit, which was designed to isolate RNA from up to 20 mg of tissue. Under the constraints of the requirements for the Maxwell RSC Blood DNA kit, we can make an informal recommendation for the isolation of DNA from mammalian tissues. We have done some work testing small pieces of mouse tissue in the Maxwell RSC Blood DNA kit and while this isolation method has not been developed as a product, we can certainly share how this was done.
- Samples were incubated in 300 µL of sterile water, plus 30 µL of Proteinase K for an hour to overnight.
- The resulting lysate was combined with 300 µL of RNA Lysis Buffer (We sell this as a standalone buffer as Cat. #Z3051), and vortexed briefly. This will result in approximately 630 µL of total lysate volume.
- All the liquid lysate was then loaded into a Maxwell RSC Blood DNA kit and isolated as per the technical manual. We could then confirm high concentration DNA as a result.
As always, if you are looking to isolate DNA or RNA from an unusual sample type, please contact the Promega Technical Services Group, and we will be happy to guide you through a solution. Read about some of the more unusual sample types we have supported in the past.
Literature Cited
- Harrington, C.A. et al. (2020) RNA-Seq of human whole blood evaluation of globin RNA depletion on Ribo-Zero library method. Nature https://www.nature.com/articles/s41598-020-62801-6
Paraj Mandrekar
Paraj Received a M.S. in Genetics, studying cancer genetics and population genetics at the University of Wisconsin-Madison before joining Promega as a Research Scientist in 1998. Over sixteen years in Promega R&D, he developed or designed many paramagnetic particle based DNA purification kits, including the chemistry used in the Maxwell® RSC Blood DNA kit, and several extraction and isolation methods for the company’s Genetic Identity product line. In 2014, Paraj left a role as a Senior Scientist in R&D and moved to Promega’s Technical Services group. He says “The common thread between R&D and Technical Services is the opportunity to learn something new and interesting every single day”. He enjoys roleplaying games, and painting miniature figures when not taking care of his two sons, Alex and Kiran, with wife Michelle, who insists that they keep cats as well.
Michelle Mandrekar
Michelle received an M.S. in Genetics at the University of Wisconsin-Madison studying Arabidopsis meristem development and aging in plants. She started working as a Research Scientist at Promega in 1997. She says, “One of the most rewarding parts of my job is seeing how the kits I design help scientists do their work and when there is a direct connection to the world.” In particular, after the attack on the World Trade Center one kit was used in the process of making genetic identity reference samples for victims. More recently, the Maxwell Viral Total Nucleic Acid Purification Kit is being used for COVID testing. She developed many nucleic acid purification kits including the Maxwell simplyRNA kits. She enjoys dancing and reading and trying to get husband Paraj and sons Alex and Kiran to do more of those too. The two cats are exempt from reading.
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