Validation of novel extraction reagents to protein analysis with dried blood spots.
1
Citation
0
Reference
20
Related Paper
Citation Trend
Abstract:
AbstractScientific methods of analysis are used to reconstruct crime scenes. However, a lack of sufficient bloodfor analysis is common at crime scenes. To apply scientific analysis methods to crime scenes, it is importantto extract components from the very small amounts of blood samples without damage. We previouslydeveloped a novel extraction reagent for analyzing bloodstains at crime scenes. The developedextraction reagent contains Tris-EDTA (TE), which preserves DNA and phosphate-buffered saline (PBS),which preserves proteins. The developed extraction reagent exhibited superior performance with respectto DNA extraction. Blood was collected from 23 healthy adult men and women in vacutainers that didnot contain coagulants or anticoagulants. These samples were used to generate DBSs under differenttemperature and humidity conditions. Proteins were extracted at five time points (day 1, 7, 14, 21, and30) using the developed extraction reagent, previously used agents TE and PBS, as well as theconventional agent double distilled water (D.D.W). The protein extraction performances of theseextraction agents were compared and analyzed using western blotting. We also quantitatively confirmedthe amount of protein extracted using commercialized GAPDH. We cross-validated the protein extractioncapability of the extraction reagent developed using two verification substances from the blood cells ofthe crime scene and confirmed that the protein extraction performance was excellent.Keywords:
Distilled water
Blood Stains
Dried blood
Phosphate buffered saline
Protein purification
Cite
To select the optimum extraction method for total RNA from peanut cotyledon,the extraction effect of modified guanidine-acid-phenol extraction method,modified CTAB method and column centrifugation extraction kit method were compared with HY22 tissue culture seedlings as materials.The results showed that the RNA extraction effect of extraction kit method could be significantly affected because the samples could always be blocked in columns.The two modified methods could avoid this phenomenon and save cost,so they had better extraction effect by the detection of UV spectrophotometer and electrophoresis.The RT-PCR results showed that the total RNA extracted by the modified methods could be directly used for molecular biology experiments,such as molecular cloning and gene expression analysis.
Cotyledon
Guanidine
Cite
Citations (0)
To explore the extract methods of Dermatophagoides pteronyssinus (D. pteronyssinus), the extracts were prepared with Coca's solution, lysis solution of two-dimensional (2D) electrophoresis and Trizol reagent respectively. In protein concentration assay, BCA reagents were used. The protein concentrations in the extracts by different methods were: lysis solution Trizol reagent Coca's solution. Two-dimensional electrophoresis was done with these different extracts. The experiment results were: 1) many protein spots at low molecular weight (MW) in extract with Coca's solution method; 2) more protein spots at low MW in extract with lysis solution method; 3) several medium MW protein spots(e. g. 174-178ku ) and more low MW protein spots in extract with Trizol reagent. There are five peculiar proteins in every 2D map in our experiment (twelve times) with Trizol reagent. There were low protein concentration and less protein spots with Coca's extract. There were few protein spots at medium MW with lysis solution though they have higher protein concentration. It was shown that Trizol reagent extracted more protein components of D. pteronyssinus PMB than that by Coca's solution and lysis solution. The five peculiar proteins in 2D map with Trizol was reagent considered as one of finger printing of D. pteronyssinus PMB in 2D electrophoresis map.
Trizol
Spots
Cite
Citations (0)
Objective In order to seek a suitable method for extraction of DNA from dried and corruption mouse samples. Methods Improved and traditional phenol-chloroform extraction, boiling method, Chelex-100 and silica beads were used to extract DNA from dry and corruption rat hair, hides and bones and other materials. The extracted DNA was amplified by PCR and sequenced. Results High-quality DNA could be extracted from hair,hides and bone samples of fresh mice by all the methods described above. Enough DNA could obtained from dried hides and corrupt bone samples by using of phenol-chloroform extraction and Chelex-100 method, respectively.Conclusion It was possible to extract high quality DNA from dried or corrupt samples by using of appropriate methods to meet the need of further researches.
Cite
Citations (0)
Abstract Abstract Common methods for the extraction of DNA from whole blood involve several phenol extraction steps. Phenol is corrosive and toxic, and the extraction steps are time-consuming, limiting the number of samples that can be processed. Two other methods using high salt or guanidine hydrochloride were tested for extraction of DNA from sheep blood. Extraction using the guanidine hydrochloride method resulted in a gelatinous material that failed to resuspend in TE buffer. The high salt method produced a good yield of high molecular weight DNA as determined by agarose gel electrophoresis. The mean yields of DNA from 100 samples of 20 ml of whole blood extracted by either the phenol or the high salt methods were 0.50 mg (SD=0.19) and 0.64 mg (SD=0.26) respectively. The quality of the DNA extracted by the high salt method was equivalent to that extracted by the phenol method and the DNA from both methods was digested to completion with a range of restriction enzymes and was suitable for analysis by Southern blotting. The high salt method can be used routinely for the extraction of DNA from sheep samples, increasing the number of samples processed and facilitating DNA extraction for genetic linkage analysis and diagnostic tests. Keywords: DNA extractionwhite blood cellssheepSouthern blotting
Agarose gel electrophoresis
Guanidine
Agarose
Hydrochloride
Cite
Citations (236)
Objective To extract high-quality total RNA from micro-tissues and to extract genomic DNA and total protein at the same time,the traditional Trizol reagent method to be modified.Methods Mouse liver total DNA,RNA and protein were extracted by modified Trizol reagent method,which was compared with RNA extracted by traditional Trizol reagent method and genomic DNA extracted by commercial kit,besides protein was also compared with that of extraction by traditional RIPA buffer lysis method.Results The yield of RNA extracted from mouse liver tissue by modified Trizol reagent was 3.64 μg /mg,while the tradition method was 1.95 μg /mg.The average D260 /D280 value was 2.1 and 2.01,respectively.The modified Trizol method could acquire more intact RNA than traditional one,besides the ratio of 28 S/18 S was more approximate to 2:1 which seen from the picture of agarose gel.Although the yield of DNA extraction by modified Trizol reagent was obviously lower than that by commercial kit,which was 0.49 μg/mg and 1.78 μg/mg,separately,purity was kept good.The average protein yield of extraction using Trizol reagent was 50%～60% of that of RIPA buffer lysis method.After the validation of SDS-PAGE electrophoresis and Coomassie blue staining,no significant protein loss was found.Conclusion Total RNA,DNA as well as protein are able to be extracted by modified Trizol reagent method simultaneously,which is more efficient and qualified in total RNA extraction than traditional Trizol method;more convenient and economical in genomic DNA extraction than commercial kit,better in preserving small protein in total protein extraction than RIPA buffer lysis method.
Trizol
Coomassie Brilliant Blue
Agarose gel electrophoresis
genomic DNA
Lysis buffer
Bradford protein assay
Cite
Citations (0)
Objective: Extracted DNA from peripheral blood of hepatitis B virus-related clinical disease is the basis of researching,the quality and quantity of DNA extracted directly related to the success of downstream research. Economic, efficient and convenient peripheral blood DNA extraction method for the study of disease at the molecular level is particularly important. Our study was designed to compare the two kinds of peripheral blood DNA extraction method, thereby providing a strong reference for clinical research.Methods: Anticoagulation was used for the test sample. Improved salting-out method and DNA extraction kit method(purification by silica gel column) were used for genomic DNA extraction respectively, by spectrophotometer meter measured the DNA concentration and purity, PCR amplification and electrophoresis experiment was carried on. Comparison of the effect of improved salting-out and DNA extraction kit. Results: Kit(purification by silica gel column), which saved time, with higher DNA-purity and better DNA single PCR bands,had little dosage of extraction specimens. Improved salting-out method had easier operation and higher DNA-yield, but the PCR band included many impurity bands. Conclusions: Both the two methods had advantages and disadvantages, kit(purification by silica gel column) was reliable and fast, but received less DNA which was degraded easily, improved salting was time-consuming, but received higher DNA concentrations and the amount. According to different clinical research needs and conditions, for example, the experimental time and money, DNA purity and concentration required for the experiment, different volumes of the sample to provide comprehensive selection of suitable extraction method.
Salting out
genomic DNA
Cite
Citations (0)
Objective To compare the different effect of rat genomic DNA extracted from whole blood by the methods of salt-extraction of high,upgrade salt-extraction of high and ultrasonic treatment. Methods 200 μl anticoagulated blood with liquemine was extracted genomic DNA by above three methods respectively. Then the genomic DNA purity and yield were measured by ultraviolet photometer and agarose gel electrophoresis. Results The genomic DNA yield extracted by the method of salt-extraction of high is 38μg/ml whole blood,the method of upgrade salt-extraction of high is 76.5μg/ml whole blood and the method of ultrasonic treatment is 20μg/mL whole blood. The DNA purity was indicated by A260/A280:the method of salt-extraction of high is 1.62; the method of upgrade salt-extraction of high is 1.46; the method of ultrasonic treatment is 4.10. Conclusions Three methods of genomic DNA extracted from whole blood were quick,safe and easy to operate. The yield of genomic DNA extracted by the method of upgrade salt-extraction of high was the highest.
genomic DNA
Agarose gel electrophoresis
Agarose
Cite
Citations (0)
Distilled water
Cite
Citations (8)
Lysis buffer
Sample Preparation
Alkaline lysis
Cite
Citations (15)
Objective To explore a simple method of DNA extraction from human blood clots. Methods Blood clots thawed in room temperature, homogenized extraction buffer including higher sodium chloride and higher EDTA, the homogenized samples were digested with digestion buffer and extracted with phenol-chloroform. Results The genomic DNA were successfully extracted. The average quantity of the extracted DNA was (32±10)mg/L,while A_ 260 /A_ 280 1.8. Conclusion The improved extraction method was reliable for obtaining high quantities of DNA from blood clot suited for PCR amplification.
genomic DNA
Human blood
Cite
Citations (0)