Chloroform water

Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. •Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.

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Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.

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Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol-chloroform-isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.

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Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10–100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources. Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10–100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources. butanol and methanol cholesteryl ester ceramide diacylglycerol diisopropyl ether free cholesterol glucosylceramide lactosylceramide lyso-PA lysophosphatidylcholine methyl-tert-butyl ether neutral loss phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol precursor ion scanning phosphatidylserine sphingomyelin triacylglycerol Lipids belong to a class of molecules with an immense structural diversity and function (1Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr, A.H. Murphy R.C. Raetz C.R. Russell D.W. Seyama Y. Shaw W. et al.A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-861Abstract Full Text Full Text PDF PubMed Scopus (1127) Google Scholar). In addition to their important role in conserving membrane integrity, they participate in many intracellular processes, such as signal transduction, apoptosis, and membrane trafficking. As a consequence, disturbances in lipid metabolism have been implicated in several diverse diseases, such as type 2 diabetes, Alzheimer disease, (2Hartmann T. Kuchenbecker J. Grimm M.O. Alzheimer's disease: the lipid connection.J. Neurochem. 2007; 103 (Suppl. 1): 159-170Crossref PubMed Scopus (161) Google Scholar) and cancer (3Ogretmen B. Hannun Y.A. Biologically active sphingolipids in cancer pathogenesis and treatment.Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1006) Google Scholar). Lipid analysis has been an important area of research for several decades, and due to technological advances, the field has experienced a renaissance in the last decade. In a modern laboratory, a comprehensive lipid characterization can be performed that generates quantitative data of several hundreds of molecular lipids from several different lipid classes. This kind of analysis, often called lipidomics, is based on HPLC and mass spectrometry (MS) instrumentation. The analysis is performed unattended in the 96-well format and is fully automated. An important component for successful analysis is the quality of the lipid extract. It is important that the lipid extract that is injected on the HPLC or infused into the mass spectrometer is pure; therefore, it is important that interfering substances and particles are removed. Inability in removing these substances might result in a high chemical background, which will have an effect on both the sensitivity and selectivity of the analysis. In contrast to fully automated and high-throughput analysis, lipid extraction is still often performed manually, involving exhaustive and time-consuming pipetting steps and hazardous solvents such as chloroform. Thus, a fully automated, chloroform-free method that can be used with standard 96-well robots would significantly improve sample throughput, as well as reduce the negative impact on health and environment. The aim of this study was to develop that method. Two of the most commonly used methods for extracting and purifying lipids are the Folch (4Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) and Bligh and Dyer (5Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar) procedures. Even though both of these methods are highly efficient for extracting lipids within a wide range of hydrophobicity, they are associated with important disadvantages. Both of these methods are based on chloroform, which in a mixture with methanol, is the primary solvent for solubilization. After separation of the lipids from nonlipids by creation of a two-phase system by adding a buffer, the lipids end up in the chloroform phase the of the This that for of the lipid the phase and the which often have to be This step can to of the extract and of the and HPLC methods free of chloroform, such as the methods by et A in plasma and the metabolism of PubMed Scopus Google Scholar) and et A solvent system for of plasma or Lipid Res. Full Text PDF PubMed Google are an phase the these methods are with the extraction of lipids with a wide range of or result in a method based on methyl-tert-butyl ether was by et Lipid extraction by methyl-tert-butyl ether for high-throughput Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). Even though this method was to have a phase and to be for total lipid the method has a high automation in the 96-well format In this we a fully automated lipid extraction in which we are to extract lipids from 96 plasma or samples in only 60 The method, which is free of is based on a mixture of butanol and methanol (BUME) for the initial extraction the method has a and it is based on solvents with the lipids will be in the their and the for to analysis. We an of the method for automated of the total lipid extract into a and a neutral methanol, chloroform, and diisopropyl ether were all of HPLC and from was from and acetic acid was from lipid were from Lipids lysophosphatidylcholine and cholesteryl ester were from and diacylglycerol were from and the free cholesterol was from for were in with the standard and they were by Glass were from Lipid was from and was from of samples were by in or samples are called and high the phase of the method human plasma samples from the were and In our to develop a and efficient automated extraction a of were for the initial one-phase extraction step which is important for the and rapid of and of were solvents and and solvents and The was that the in with or a one-phase mixture with or plasma a less After 300 µl of solvent with µl the mixture was and the automation in The solvents for the two-phase extraction for separation and purification of the lipid were and or acetate or in with the were that addition of 300 µl solvent 2 and 300 µl acetic acid as to the one-phase system would result in a of a two-phase system to the in in with recoveries of a lipid and of nonlipids in the the of solvent for of an method. This method the basis for the BUME for the of the automated extraction µl plasma in 96-well one-phase extraction with a efficient and and of in the initial one-phase two-phase extraction with an phase of total of and within phase automated steps for and to automated extraction extraction 96-well efficient and and in a After the of extraction solvents using 1% acetic acid as buffer, a was used for of the of the The that were were the of acetic acid or in the phase and the of methanol in solvent and acetate in solvent 2 The was performed using After the protocol in of solvent and the method was up on a pipetting such as and of the different and were to high recoveries and a protocol of extracting sample from µl the protocol The of the automated BUME method was a performed Folch method in of lipid class of molecular in several lipid classes, and the to extract samples in The automated BUME method was for sample range and on health and were for the BUME method the Folch method. Lipids were extracted using a Folch (4Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) commonly used in our µl methanol was to 60 µl Lipid in a After of µl chloroform was After of 300 µl of acetic acid was and the two-phase system was for After of the phase was to a and the phase was with µl chloroform. of and of to phase the were and in The lipid were analysis. Lipid of Lipid with and were to a of in and injected into a HPLC and were and using to separation and of neutral and lipid classes by and mass PubMed Scopus Google Scholar). The HPLC system was used in with a for purification of and ceramide for analysis. of phosphatidylcholine sphingomyelin and was performed using and total lipid were in with acetate with The were infused into a with a ion The was for precursor ion scanning of and in ion to E. J. and of molecular by precursor ion Chem. PubMed Scopus Google of by precursor ion scanning on a mass Chem. PubMed Scopus Google Scholar). After purification using the HPLC the were in with acetate as This was analyzed using the and the were in by neutral loss of the most R.C. J. E. of the of diacylglycerol and triacylglycerol molecular in using neutral loss mass Biochem. 2007; PubMed Scopus Google Scholar). as a lipid class using the HPLC profiles of the molecular were obtained from the total lipid extract using the the was for of in ion M. T. B. of cholesterol and cholesteryl ester by mass spectrometry PubMed Scopus Google Scholar). data from the was using the The obtained from the HPLC purification were analyzed using HPLC to a mass spectrometer as M. M. B. J. is associated with in the lipid and of from with type 2 PubMed Scopus Google Scholar). An of the absolute recoveries of neutral lipids was by 60 µl of Lipid with and After BUME the was in the As a reference 60 µl of Lipid was and of was into the lipid extract to The was using a recoveries of neutral lipids using the BUME cholesteryl ester triacylglycerol diacylglycerol are in a are of absolute recoveries of and a of molecular lipids from different lipid classes were into 60 µl of Lipid and extracted using both the Folch and BUME analysis, of molecular lipids was which was used for As a reference 60 µl of Lipid was extracted and with both of of the lipid was using on the of the lipids was using and for recoveries of and was the and standard 2 was the As a reference both were the was the and standard 2 was the As a reference both were the and sphingolipids were using precursor ion scanning and neutral loss a for of Lipid was used as sample are was the and standard 2 was the As a reference both were the and sphingolipids were using precursor ion scanning and neutral loss a for in a of Lipid was used as sample are were performed using was solvents were to they the automation in of the solvent for butanol:methanol (BUME) for the initial one-phase a mixture of butanol:methanol to was to plasma in the 96-well a one-phase system with a that was to the BUME as solvent well in with solvent 2 and or acetate or in with and the and all in for the initial it that the of solvent to is important to the of a in the initial one-phase We investigated the of of or chloroform to a one-phase The data that the of methanol with of the solvent methanol was for of butanol:methanol to with but almost was for of solvent 2 and to plasma with BUME in a rapid and phase separation was used or in with acetate or phase separation within with a The of these solvents in a lipid extract that efficient to and of of the phase and phase that and methanol were in the phase and that butanol and solvent 2 were in the of the of solvent 2 as as was the of methanol in butanol BUME the of acetate in and the of or acetic acid in the buffer, a was This that for a BUME a of methanol in butanol the recoveries of As in using methanol in butanol in a for with a methanol was used in the BUME The solvent that the of solvent 2 was less the recoveries of was from less with to almost with acetate the acetate the lipid but the of from 1% with acetate to with acetate (3:1) was for the BUME protocol. The from the that was as were only with to lipid recoveries the acetic acid in was a high in as well as a be for the extraction of lipids that be to as the high of acetic acid might have on lipid for the we this by the to a Lipid sample and extracting it with or 1% acetic The result in the extraction the different and we the that is by the acetic acid data are by the high absolute extraction for the using the BUME to the BUME as solvent a mixture with plasma that was the in contrast to the for a mixture of as solvent result in phase was included in solvent and therefore, it from and and automation and less The that the of was only with as solvent and as solvent similar to the for BUME with methanol in butanol The of and of methanol in the initial one-phase extraction in recoveries of or to the of the in as an to in solvent 2 different from or better A protocol was based on the automation and solvents and This which is in was developed on a the protocol can be to standard 96-well that can with µl with for 96-well lipid solvent solvent buffer, sample and solvent A the is in The total is 60 the method is fully automated, only of is for 96-well This that a samples can be extracted with only of only 60 samples can be extracted in our using the Folch the BUME and Folch lipids were in samples with of triacylglycerol this 60 µl of Lipid or was extracted and high and lipids were As can be in the extraction methods in recoveries for all high plasma lipids and as well as for less biologically important lipids and The only the methods was an in the extraction for in the and samples using the BUME using of the total lipid extract on the we profiles of molecular lipids for several lipid classes. profiles of molecular lipids from lipid classes from the The profiles for the automated BUME and Folch that the automated BUME method profiles of molecular lipids in both the and of the An of absolute extraction was by using a of and lipid and lipids were as the was the of the signal was from the lipid The high extraction of the neutral lipids and for the BUME method these we the absolute using the Folch from the lipids in the BUME and Folch extraction The high and extraction for the sphingolipids and and phosphatidylethanolamine with absolute recoveries for both the BUME and Folch In for the phosphatidylserine and phosphatidylglycerol the BUME method was phosphatidic acid which is the most both the BUME and Folch extraction The most the methods was for the lyso-PA lipid class in which Folch a with for the BUME In addition to the we used to the of the methods to substances in the Both the Folch and BUME in an of the in the were the methods in of ion or using and from both methods well with the In these similar extraction for BUME and Folch with significantly recoveries for the most lipids with the BUME An important step in the lipid extraction is the initial one-phase extraction in in this a butanol:methanol (3:1) mixture was to the sample A might to the of a two-phase which will result in extraction the of sample for we extracted different of Lipid of The that the automated BUME method is linear extracting 10–100 µl to The and were investigated in this based on their and high recoveries similar can be for these lipid classes. The BUME method less in of extraction and the of for lipid research. to the on and as well as the to chloroform Folch extraction is to an environmental solvents replace chloroform, and of solvents are used the novel BUME method a step in a modern lipid laboratory in of and environment. In addition to the BUME method, we developed an automated extraction method for the separation of neutral and lipid classes. This extraction with a total lipid extract to which µl of is The is using the for min, and 300 µl and 300 µl is The 1% of After and phase the phase the neutral lipids is and the methanol phase is with 300 µl of of this extraction method that of the neutral and lipid classes up in the of the and lipid class up in the methanol The data that profiles of lipid were this separation The the separation into the on the acid the was in in the the was in the phase In this we developed a novel and fully automated method for total lipid extraction from plasma or using a The method, which is based on is performed in 60 It almost extraction with the standard methods, with with to sample and as well as and environmental the field of lipid analysis, extraction is an often but highly important The of automated lipid extraction can and to on data and analysis. as extraction is often associated with pipetting and of automation has health extraction based on the Folch have been by and have been developed in our T. quantitative molecular PubMed Scopus Google M. J. J. by mass PubMed Scopus Google Scholar). such as a high a and the for in a This with environmental to the to develop a method based on which in the BUME method. In both the Folch and the Bligh and Dyer chloroform is used with methanol for the initial one-phase and of the sample This step is important for and critical for we and a of butanol and methanol for this to chloroform, butanol lipids and is in with methanol, and the to as well as the of butanol and methanol for one-phase lipid extraction from We that the of as a result of the addition of the BUME mixture in a that was to using the automated As this was the BUME mixture was to the plasma samples to extraction can be This is as it might reduce the of is an often in with for the initial one-phase extraction A in plasma and the metabolism of PubMed Scopus Google Lipid extraction of with a Biochem. PubMed Scopus Google Scholar). as we method the of this the lipid phase and the phase was to the and of the solvents and In we from the and of the lipid and that the BUME of the of the into in a phase and a lipid The of the BUME mixture to in this the two-phase extraction step is an important for high lipid and of the in contrast to or chloroform and only a of methanol is to butanol in the of less The phase highly phase only a of methanol and This the recoveries of lipids using solvent The of the addition of the solvent mixture is to extracted from the sample by the BUME from nonlipids in the phase by of a two-phase The of lipids the lipid phase and the phase is by the of the lipid phase to both and lipids This a lipid phase with a high of the phase be as as with a high of to only such as and We that a mixture with in high recoveries of all investigated lipids but that addition of acetate to was in for In the method we used a with a standard µl and a standard fully in the and the samples and solvents as as a and the such a by and The were and steps was used only adding 300 µl BUME to the plasma is the method of are in the The is µl of the of the lipid extract the and it the of the phase a both in the and in the the solvent is a into the and a for efficient two-phase lipid on the used and the of and the be up the BUME protocol. a of the phase is therefore, performed only automated several steps can be In the BUME method, we the phase and the are and This to high extraction the extraction in a the developed method, we to the Folch method, which is of the methods in the we lipids in samples with of The addition of was a the Folch and Bligh and Dyer that a high of can result in loss of and extraction of the Bligh and Dyer and Folch methods for total lipid in a range of PubMed Scopus Google Scholar). almost extraction recoveries the BUME and Folch of the for all lipid classes, with the of This lipid was less extracted using the BUME method. the in extraction the Folch and BUME was using it was using which be by the that result from of the abundant sample which is the for The Folch protocol is to as it and is performed a the BUME In the BUME solvents are from the and are used with We our to Folch by the extraction for the developed BUME method for a of different lipid classes. Both the Folch and BUME methods high and similar recoveries for the neutral and the BUME better recoveries for the lipids and It has been that the Folch is for extracting and that the addition of acetic acid can be used to the recoveries of lipids in extraction A of extraction methods for the isolation of from biological Biochem. PubMed Scopus Google of by Biochem. PubMed Scopus Google Scholar). using a Folch protocol of acetic the BUME protocol better recoveries of the It might be that the of acetic acid in the BUME with for the Folch to BUME recoveries of The of this lipid class using the Folch has been and several the of butanol for extraction of this lipid class M. analysis of and acid in plasma samples by mass Chem. PubMed Scopus Google B. quantitative analysis of acid molecular by Biochem. PubMed Scopus Google Scholar). An important with the BUME method is We the of the method and that efficient and extraction recoveries be obtained µl of sample in the initial one-phase This is with the Folch method that a of which the sample compatible with the automated a chloroform-free method using a of and methanol was Lipid extraction by methyl-tert-butyl ether for high-throughput Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). The that by using this solvent for the initial one-phase high recoveries of a wide range of lipid classes be This method to be a to the Folch method for A of this method, was the of a high the initial one-phase which automation in a 96-well format the solvent phase of the of the BUME method, we that for the initial one-phase extraction step almost methanol in to less We that phase separation and was was used as the solvent for the two-phase only of was due to the of methanol with an automated method. is an field with the of a of lipids from a such a characterization lipids with a of and it is to a method for extraction of all lipids of several will have to be in or in et analyzed the E. analysis of the by quantitative mass PubMed Scopus Google Scholar). As this characterization lipids with a wide range of they performed an initial chloroform-based extraction of the neutral lipids and a of the phase to the can be for extraction of the as by et E. S. Merrill Jr, A.H. analysis of sphingolipids for using and linear ion mass Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). these automated are to reduce time-consuming extraction procedures. this we in addition to the BUME method, an automated method for the lipid extract from the BUME into and This of up and the sample is highly analysis of lipid the in were to be it would be highly to the of the to analysis. This would reduce of the ion and reduce ion which would reduce the sensitivity of the analysis. In this we have on we that the BUME method might be to extraction of highly lipids such as and in the these lipid classes are using a one-phase extraction E. S. Merrill Jr, A.H. analysis of sphingolipids for using and linear ion mass Lipid Res. Full Text Full Text PDF PubMed Scopus Google S. A for the purification and of PubMed Scopus Google Scholar). extraction of these lipid classes might be by using the initial one-phase extract for analysis the two-phase extraction step or by adding extraction steps on of the BUME method. It well be to a solvent mixture based on a high of butanol for automated of lipids in the phase from the BUME method, similar to what has been J. A.H. phase extraction and 2 of neutral and neutral Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). the of a method for the extraction of lipids the of this and to be investigated In this we have on the extraction of plasma of the was to the into This has and based on the BUME protocol have data In we a novel and fully automated method for total lipid extraction from blood plasma or We have the of using automated extraction which as well as reduce on both environmental and a for the extraction often for using a the method solvents and has been to have high extraction recoveries for all the investigated we that this method replace the chloroform-based methods often used and that the BUME method the method of for the modern lipid

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