Optimizing the immobilization of single-stranded DNA onto glass beads

The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo(1), onto glass beads via a 5'-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo(1) concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89%. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 microg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo(2), was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.     

Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study

Antibody microarrays have the potential to revolutionize protein diagnostics. The major problems in the fabrication of antibody arrays, however, concern the reproducibility and homogeneity of the attachment of the proteins on the solid substrate. We here compare the DNA-directed immobilization (DDI) method with two conventional strategies for immobilization of antibodies on glass substrates. DDI is based on the self-assembly of semisynthetic DNA-streptavidin conjugates which converts an array of DNA oligomers into an antibody microarray. DDI was compared with direct spotting of antibodies on chemically activated glass slides and with immobilization of biotinylated antibodies on streptavidin-coated slides. The immobilized antibodies were used as capture reagents in a two-sided (sandwich) immunoassay for the quantification of rabbit IgG as a model antigen. Detection limits down to 0.001nM (150 pg/mL) were attained with all three array formats; however, DDI and direct spotting of the antibodies led to the highest fluorescence intensities. DDI led to the best spot homogeneity and intra- and interexperimental reproducibility. Moreover, DDI allowed highly economical use of antibody materials; that is, at least 100-fold less antibody is needed for preparing an array by DDI instead of by direct spotting. Taking into account the greater versatility and convenience of handling of the self-assembly approach, this study demonstrates that DDI is an advantageous alternative for generating versatile and robust protein arrays.     

Improving the yield and quality of DNA isolated from white-rot fungi

A new simple method used to eliminate polysaccharides that cause problems during DNA isolation was established for 6 different white-rot fungi using 1% hexadecyltrimethylammonium bromide (CTAB) as wash buffer and followed by centrifugation. Variation in the DNA yield and quality was ascertained using precipitating agents, detergents and cell-wall-hydrolyzing chitinase. Considerable amount of exopolysaccharides from fungal biomass was removed with the use of 1% CTAB wash buffer followed by centrifugation. The DNA varied in terms of yield and quality. For the DNA extraction use of 2% SDS in extraction buffer worked best for Pycnoporus cinnabarinus, Cyathus bulleri, Cyathus striatus and Cyathus stercoreus, while 2% CTAB worked best for Phanerochaete chrysosporium and Pleurotus ostreatus. Elimination of phenol and use of absolute ethanol for precipitating DNA resulted in good yield and quality of DNA. This DNA was amenable to restriction endonuclease digestion.     

Optimizing stringency for expression microarrays

While several studies have reported methods to optimize expression microarray protocols, none have dealt directly with hybridization wash stringency. We designed a series of experiments to determine the optimal stringency conditions for microarray experiments, using reproducibility and magnitudes of log2 (test/reference) ratio values as measures of quality. Low-stringency wash conditions of cell line hybridizations led to nonspecific binding, resulting in increased intensities, decreased magnitude of ratios, and poor reproducibility. Relatively high-stringency wash conditions were found to give the best reproducibility and large magnitude ratio changes, although increasing the stringency beyond this point led to lower magnitude ratios and poorer reproducibility. The expression levels of the ERBB2 oncogene in the BT474 versus MCF7 cell lines showed that high-stringency wash conditions gave the best agreement with real-time quantitative PCR, although the magnitude of the changes by microarray was smaller than for real-time quantitative PCR. Analysis of a series of cell lines washed at the optimized stringency indicated that the rank order of relative expression levels for ERBB2 microarray clones agreed well with the rank order of ERBB2 levels, as measured by quantitative PCR. These results indicate that the optimization of stringency conditions will improve microarray reproducibility and give more representative expression values.     

Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).     

Sodium dodecyl sulfate solution is an effective between-run rinse for capillary electrophoresis of samples in biological matrices

It is common practice in capillary electrophoresis to perform some sort of capillary washing step(s) between separations. In many analyses little consideration is given to optimization of the wash, and typically a rather standard washing procedure is used involving a few minutes wash with 0.1M NaOH followed by a few minutes reconditioning with the run buffer. As an alternative to this procedure, we have investigated the use of wash solutions containing sodium dodecyl sulfate (SDS). This type of wash has been used in the analyses of both small molecules and proteins, with encouraging results. After the SDS wash, the electroosmotic flow has been shown to be restored to values close to normal in a capillary which had previously been coated with plasma proteins. Separation efficiency for a test compound (dextromethorphan) is improved if an SDS rather than a HCl---NaOH wash is used after injection of plasma. In a direct-injection analysis of plasma proteins using a pH 10 borate buffer, an SDS-based washing procedure (total time, 1 min) gave better migration-time reproducibility than an NaOH-based wash, which took 5 min in total.     

植物病毒检测芯片的杂交条件优化

利用芯片点样仪将5种侵染马铃薯的病毒/类病毒(苜蓿花叶病毒、黄瓜花叶病毒、黄瓜花叶病毒-卫星病毒、马铃薯病毒Y、马铃薯块茎纺锤状类病毒)的保守区寡核苷酸(Oligonucleotide,oligo)探针和PCR探针点样于玻片,并以植物18S rRNA作为内参照制成基因芯片。研究探针浓度、杂交时间、杂交温度以及点样液对芯片杂交的影响,并验证优化后病毒检测芯片的特异性。结果表明,寡核苷酸探针浓度介于5-20 ?mol/L之间对杂交信号强度影响不大,PCR探针浓度与杂交信号强度间呈线性关系;在45℃杂交4 h时,芯片的杂交信号最强,且该条件下进行杂交对两种探针芯片的影响趋势一致;点样液中以DMSO的杂交效果最好。经过整体条件优化后的两种探针芯片在杂交检测上具有较高的特异性,适于检测植物病毒。     

Improving protein array performance: focus on washing and storage conditions

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.     

Validated micellar electrokinetic capillary chromatography method for quality control of the drug substances hydrochlorothiazide and chlorothiazide

A stability-indicating, quality control analysis method was developed and validated for the diuretic drug substances hydrochlorothiazide (HCTZ) and chlorothiazide (CTZ). Micellar electrokinetic capillary chromatography employing the anionic detergent sodium dodecyl sulfate at 30 mM in 20 mM sodium borate buffer pH 9.5 was utilized to separate and quantify the active drug substance HCTZ from CTZ and the common impurity, 4-amino-6-chloro-1,3-benzenedisulfonamide (DSA). A 100 μm I.D. uncoated fused-silica capillary was necessary to provide the sensitivity, i.e. 1 μg/ml, for quantification of the DSA impurity. In this study, the linearity, precision, selectivity, accuracy, reproducibility and limit of quantitation for the method were investigated for HCTZ, CTZ and DSA. As the first validation of a drug substance method by capillary electrophoresis in this laboratory, unusual care was taken to insure reliability and ruggedness with multiple instruments, capillaries and analysts. Precision and reproducibility in the range of 1% R.S.D. was achieved by controlling subtle injection factors. These included minimizing the time in which the capillary ends were not immersed in buffer or sample during the injection process and minimizing the number of assays for each anode or inlet buffer vial. Stacking induced by differences in ionic strength between sample and capillary buffer was reduced by using a sample buffer concentration similar to that of the capillary buffer. Although stacking accomplished by using lower sample buffer concentrations increased sensitivity, reproducibility was decreased. Achievement of the 1% R.S.D. precision level means that many quality control assays for drugs with good absorbance characteristics can be validated with HPLC reproducibility and CE efficiency. These micellar electrokinetic capillary chromatography methods conform to the USA and European Pharmacopoeial validation guidelines.     

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Reproducibility of Circulating MicroRNAs in Stored Plasma Samples

Background

Most studies of microRNA (miRNA) and disease have examined tissue-specific expression in limited numbers of samples. The presence of circulating miRNAs in plasma samples provides the opportunity to examine prospective associations between miRNA expression and disease in initially healthy individuals. However, little data exist on the reproducibility of miRNAs in stored plasma.

Methods

We used Real-Time PCR to measure 61 pre-selected microRNA candidates in stored plasma. Coefficients of variation (CVs) were used to assess inter-assay reliability (n = 15) and within-person stability over one year (n = 80). Intraclass correlation coefficients (ICCs) and polychoric correlation coefficients were used to assess within-person stability and delayed processing reproducibility (whole blood stored at 4°C for 0, 24 and 48 hours; n = 12 samples).

Results

Of 61 selected miRNAs, 23 were detected in at least 50% of samples and had average CVs below 20% for inter-assay reproducibility and 31 for delayed processing reproducibility. Ten miRNAs were detected in at least 50% of samples, had average CVs below 20% and had ICCs above 0.4 for within-person stability over 1–2 years, six of which satisfied criteria for both interassay reproducibility and short-term within-person stability (miR-17-5p, -191-5p, -26a-5p, -27b-3p, -320a, and -375) and two all three types of reproducibility (miR-27b-3p and -26a-5p). However, many miRNAs with acceptable average CVs had high maximum CVs, most had low expression levels, and several had low ICCs with delayed processing.

Conclusions

About a tenth of miRNAs plausibly related to chronic disease were reliably detected in stored samples of healthy adults.     

Quantitative analysis of steroid profiles from urine by capillary gas chromatography : I. Accuracy and reproducibility of the sample preparation

A method is described for the determination of steroid profiles from urine by means of gas chromatography using high-efficiency glass capillary columns. The accuracy and reproducibility of essential steps in the sample preparation (extraction of steroids and steroid conjugates by means of XAD-2, enzymatic hydrolysis with Helix pomatia juice, solvolysis in acidified ethyl acetate and alkali wash) are established using different endogenously labelled urine samples, obtained from normal subjects to whom labelled steroids had been administered. Preliminary results are given on the reproducibility of the derivatization procedure (formation of methoxime-trimethylsilyl (MO-TMS) ethers), the gas chromatographic analysis and the whole method. Two procedures for the purification of MO-TMS steroid derivatives are compared. Application of the method to urine samples of patients with various endocrine disorders is included.     

Necessary conditions for isolation of tightly coupled higher plant mitochondria

A number of parameters associated with the isolation of plant mitochondria were carefully examined in the hope of optimizing the isolation technique. Dark-grown mung bean (Phaseolus aureus var. Jumbo) hypocotyls were used as the source of mitochondria. Necessary conditions for the isolation of tightly coupled plant mitochondria include: (a) gentle tissue disruption; (b) rigorous exclusion of contaminating particles from the mitochondrial fraction; (c) the use of an optimal grinding medium containing a high concentration of mannitol, 10 mm phosphate or triethanolamine buffer to maintain the pH above 7.2, ethylenediaminetetraacetic acid at 1 mm level, cysteine at a low concentration (0-5 mm), and a high concentration of bovine serum albumin (0.1-1.0%); and (d) the use of the proper wash and suspending medium consisting of mannitol, buffer, and bovine serum albumin at the same concentration as in the grinding medium.     

Real-time PCR method using capturing oligo-immobilized PCR tubes to determine the specific gene for soybean and genetically modified soybean in food matrices

A new real-time PCR method using capturing oligo-immobilized PCR tubes is described. This method was used to detect specific genes for soybean and genetically modified (GM) soybean in food matrices. In a standard reaction using soybean genomic DNA and a capturing oligo for the lectin gene (Le1) immobilized on the tube, we examined the effects of such hybridization conditions as the location, length, and amount of the capturing oligo, and the incubation time and temperature. Under optimized conditions, the copy number of Le1 was determined in a concentration-dependent manner from soybean genomic DNA and soybean lysate (DNA 10-1000 ng, r=0.99; lysate 1-100%, r=0.99). The copy number of a Roundup Ready soybean (RRS) gene was also successfully detected in a concentration-dependent manner (1-100%, r=0.99) from GM soybean lysate, using PCR tubes with an immobilized capturing oligo for the transgene. Our data indicate that this is a rapid and simple method to determine specific genes for soybean and GM soybean in food matrices.     

A simple method for removing the luminal epithelium of the mouse uterus for biochemical studies.

The luminal epithelium of the mouse uterus was removed simply and rapidly by opening the uterine horns longitudinally and 'vortexing' them in buffer with glass balls. This procedure, which requires no specialized apparatus, was developed for use with uteri in different hormonal states, and 2 uterine horns/tube, 5 glass balls and vortexing for 2 min are suggested for routine use. Under these conditions, 79--100% of the cells were removed, yielding epithelial fractions of 65--90% purity. The suspension of cell constituents recovered contained intact nuclei which could be further purified. Extensive histological examination demonstrated that the other uterine tissues were minimally damaged.     

Performance of Parasitological and Molecular Techniques for the Diagnosis and Surveillance of Gambiense Sleeping Sickness

Objectives

Recently, improvements have been made to diagnostics for gambiense sleeping sickness control but their performance remains poorly documented and may depend on specimen processing prior to examination. In a prospective study in the Democratic Republic of the Congo, we compared the diagnostic performance of several parasite detection techniques, immune trypanolysis and of m18S PCR on whole blood stored in a stabilisation buffer or dried on filter paper.

Methods

Individuals with CATT whole blood (WB) titer ≥1∶4 or with clinical signs indicative for sleeping sickness were examined for presence of trypanosomes in lymph node aspirate (LNA) and/or in blood. Blood was examined with Capillary Centrifugation Technique (CTC), mini-Anion Exchange Centrifugation Technique (mAECT) and mAECT on buffy coat (BC). PCR was performed on whole blood (i) stored in guanidine hydrochloride EDTA (GE) stabilisation buffer and (ii) dried on filter paper, and repeatability and reproducibility were assessed. Immune trypanolysis (TL) was performed on plasma.

Results

A total of 237 persons were included. Among 143 parasitologically confirmed cases, 85.3% had a CATT-WB titre of ≥1/8, 39.2% were positive in LNA, 47.5% in CTC, 80.4% in mAECT-WB, 90.9% in mAECT-BC, 95.1% in TL and up to 89.5% in PCR on GE-stabilised blood. PCR on GE-stabilised blood showed highest repeatability (87.8%) and inter-laboratory reproducibility (86.9%). Of the 94 non-confirmed suspects, respectively 39.4% and 23.4% were TL or PCR positive. Suboptimal specificity of PCR and TL was also suggested by latent class analysis.

Conclusion

The combination of LNA examination with mAECT-BC offered excellent diagnostic sensitivity. For PCR, storage of blood in stabilisation buffer is to be preferred over filter paper. TL as well as PCR are useful for remote diagnosis but are not more sensitive than mAECT-BC. For TL and PCR, the specificity, and thus usefulness for management of non-confirmed suspects remain to be determined.     

Modification of Oligo (Poly) Nucleotide Phosphomonoester Groups in Aqueous Solutions

Abstract

Selective modification of oligo (poly) nucleotide phosphomonoester groups in an aqueous medium by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide in the presence of various nucleophilic agents has been investigated. Optimal conditions of the modification by amino- and hydroxycompounds have been found. Based on these studies a general efficient method for preparation of oligo (poly) nucleotide phosphoamidates and phosphodiesters in an aqueous solution has been developed. The method allows to prepare both oligodeoxyribonucleotide derivatives at 3′- and 5′-terminal phosphate groups and oligoribonucleotide derivatives at 5′-terminal phosphate groups with 80–100% yields.