Quantitation of Plasma Renin Activity in Plasma Using Liquid Chromatography & ndash;Tandem Mass Spectrometry (LC-MS/MS)
We describe a semi-automated and simple method for the accurate and precise measurement of PRA from 500 & mu;L of plasma (250 & mu;L if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37 & deg;C in buffering conditions at pH 6, the reaction is quenched with 10 % formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. ...
Source: Springer protocols feed by Imaging/Radiology - January 7, 2016 Category: Radiology Source Type: news

Quantification of & gamma;-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry
We describe a simple stable isotope dilution method for accurate and precise measurement of & gamma;-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 & mu;L) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and inject...
Source: Springer protocols feed by Imaging/Radiology - January 7, 2016 Category: Radiology Source Type: news

Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 56 & trade; Mass Spectrometer
Triosephosphate isomerase (TPI) is a glycolytic enzyme which catalyzes the interconversion between glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). TPI deficiency results in accumulation of DHAP in human red blood cells and other tissues. The disease is characterized by congenital hemolytic anemia, and progressive neuromuscular dysfunction. The laboratory diagnosis is generally made by measurement of TPI activity in RBCs. Measurement of DHAP can be useful in further confirmation and follow-up of the disease. We developed HPLC/TOF-MS method for quantitation of DHAP in RBCs. The method involves simple ...
Source: Springer protocols feed by Imaging/Radiology - January 7, 2016 Category: Radiology Source Type: news

High-Throughput Serum 25-Hydroxy Vitamin D Testing with Automated Sample Preparation
Serum from bar-coded tubes, and then internal standard, are pipetted to 96-well plates with an 8-channel automated liquid handler (ALH). The first precipitation reagent (methanol:ZnSO4) is added and mixed with the 8-channel ALH. A second protein precipitating agent, 1 % formic acid in acetonitrile, is added and mixed with a 96-channel ALH. After a 4-min delay for larger precipitates to settle to the bottom of the plate, the upper 36 % of the precipitate/supernatant mix is transferred with the 96-channel ALH to a Sigma Hybrid SPE® plate and vacuumed through for removal of phospholipids and precipitated proteins. The fil...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of 1,25-Dihydroxyvitamin D2 and D3 in Serum Using Liquid Chromatography-Tandem Mass Spectrometry
We present here an effective and accurate method for measuring 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) after immunoaffinity extraction. The MS/MS is operated in multiple reaction mode with positive electrospray. Quantification is based on peak area ratios of the analytes to respective deuterated internal standards. This method offered a linear range from 4.0 to 160.0 pg/mL with analytical recovery of 89.9–115.5 % for both 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Urinary Succinylacetone Analysis by Gas Chromatography-Mass Spectrometry (GC-MS)
Succinylacetone (SA) is used for the diagnosis and monitoring of patients with tyrosinemia type I (Tyr I). SA is exclusively elevated in blood and urine of patients with Tyr I. As urinary SA concentration is much higher than blood, SA is usually tested in urine samples. Urinary SA quantitation by gas chromatography mass spectrometry (GC-MS) is described in this chapter. The urine sample in the amount of 1 μmol creatinine is used for testing. 3,4,5,6,7-13C5-succinylacetone (13C5-SA) is used as an internal standard (IS). SA and 13C5-SA are oximated and extracted from urine with organic solvents, and then derivatized to fo...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of Free Carnitine and Acylcarnitines in Plasma or Serum Using HPLC/MS/MS
Acylcarnitines are formed by esterification between fatty acids CoA or organic acids CoA molecules and carnitine. In various fatty acids oxidation defects and organic acidurias, there is increased concentration of corresponding acylcarnitines. Abnormalities in specific acylcarnitines are used in the diagnosis of fatty acids oxidation defects and organic acidurias. Most commonly used method for the assay of acylcarnitines is HPLC-tandem mass spectrometry (HPLC/MS/MS). A HPLC/MS/MS method is described for the quantification of number of acylcarnitines. The method involves butylation of carnitine/acylcarnitines using acidifie...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone by Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS)
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders due to enzymatic defects in the biosynthetic pathway of cortisol and/or aldosterone. The analysis of cortisol, 17-hydroxyprogesterone (OHPG), dehydroepiandrosterone (DHEA), 11-deoxycortisol, and testosterone is generally performed in the diagnosis and/or follow-up of CAH. Cortisol is generally analyzed by immunoassays whereas other hormones are preferably assayed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). A multiple reaction monitoring, positive mode atmospheric pressure chemical ionization, LC/MS/MS method is described for ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

A Simple, High-Throughput Method for Analysis of Ceramide, Glucosylceramide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS
A unique monophasic extraction system coupled with LC/MS/MS to reduce matrix effects for sphingolipid analysis was developed. A solvent mixture of methanol, acetonitrile, and water was identified to simultaneously extract multiple sphingolipids with broad polarity range. To reduce matrix effects, the targeted sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The extraction solvent was used as an isocratic mobile phase in chromatographic separation to eliminate solvent exchange steps and enable high-throughput multiple lipid assay. The assay is linear for ceramide from 0.6 to 9 μg/mL with bias
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry
We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a diagnostic test. SAM and SAH are key metabolic intermediates of methionine metabolism and the methylation cycle. Determination of SAM and SAH in plasma was performed by high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards (2H3-SAM and 2H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 μL samp...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Plasma Renin Activity in Plasma Using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS)
We describe a semi-automated and simple method for the accurate and precise measurement of PRA from 500 μL of plasma (250 μL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37 °C in buffering conditions at pH 6, the reaction is quenched with 10 % formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. Quantitation is...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Urine Pyrimidine Metabolite Determination by HPLC Tandem Mass Spectrometry
Pyrimidine diseases result from deficiencies in pyrimidine de novo synthesis, degradation, and salvage pathways. Enzymatic deficiencies in pyrimidine catabolism lead to mitochondrial neurogastrointestinal encephalopathy (MNGIE), pyrimidinuria, dihydropyrimidinuria, ureidopropionic aciduria, and other disorders. While MNGIE presents with gastrointestinal dysmotility, cachexia, and leukoencephalopathy, pyrimidinuria and dihydropyrimidinuria may show symptoms of epilepsy, autism, mental retardation, and dysmorphic features. The application of HPLC-MS/MS facilitates rapid screening of pyrimidine metabolites. Here we describe a...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Urine Purine Metabolite Determination by UPLC-Tandem Mass Spectrometry
Inborn errors of purine metabolism, either deficiencies of synthesis or catabolism pathways, lead to a wide spectrum of clinical presentations: urolithiasis (adenine phosphoribosyltransferase), primary immune deficiency (adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency), severe intellectual disability, and other neurological symptoms (Lesch-Nyhan disease, adenylosuccinase deficiency, and molybdenum cofactor deficiency). A rapid quantitative purine assay was developed using UPLC-MS/MS to determine purine nucleoside and base concentrations in urine. Taking advantages of ultra performance liquid c...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Determination of Phenylalanine and Tyrosine by High Performance Liquid Chromatography-Tandem Mass Spectrometry
Hyperphenylalaninemia/phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism affecting about 1:15,000 infants in the United States. PKU is an autosomal recessive disorder that if untreated results in mental retardation. The most common cause of PKU is deficiency of the enzyme phenylalanine hydroxylase that converts phenylalanine to tyrosine. Tyrosine deficiency results in impaired synthesis of catecholamines and thyroxine. Less commonly, it can result from defects in the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor for the enzyme phenylalanine hydroxylase. In...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Parathyroid Hormone in Serum or Plasma by Liquid Chromatography-Tandem Mass Spectrometry
Parathyroid hormone (PTH), an 84 amino acid peptide hormone, is an important regulator of calcium homeostasis. Quantitation of PTH in serum is useful for the diagnosis of primary hyperparathyroidism, hypoparathyroidism, and for monitoring osteodystrophy in patients with renal failure. The biological activity of PTH arises from binding of PTH (N terminus) to its target receptor (D’Amour et al., Kidney Int 68: 998–1007, 2005). Several C-terminal and N-terminal fragments circulate in normal subjects. Recent studies have demonstrated that accurate quantitation of PTH fragments may be of clinical value. In this chap...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

High Sensitivity Measurement of Pancreatic Polypeptide and Its Variant in Serum and Plasma by LC-MS/MS
Aliquots of serum or plasma samples are combined with stable isotope labeled internal standard. Pancreatic polypeptide (PP) and its truncated variant PP3-36 are enriched by incubation with anti-PP antibody conjugated to magnetic beads. Peptides are eluted from beads in acidic buffer and the samples analyzed using liquid chromatography coupled with tandem mass spectrometry. Instrumental analysis of PP and PP3-36 is performed using electrospray ionization ESI in positive ion mode and multiple reaction monitoring (MRM) acquisition. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitative Organic Acids in Urine by Two Dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS)
Seventy-six organic acids in urine specimens are determined with quantitative two dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS). The specimen is treated with urease to remove urea then derivatized to form pentafluorobenzyl oximes (PFBO) of oxoacids. The sample is then treated with ethyl alcohol to precipitate proteins and centrifuged. After drying the supernatant, the organic acids are derivatized to form volatile trimethylsilyl (TMS) derivatives for separation by capillary two dimensional Gas Chromatography (GCxGC) with temperature programming and modulation. Detection is by Time of Flight ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Mass Spectrometry in Clinical Laboratory: Applications in Biomolecular Analysis
Mass spectrometry (MS) is a technique that can identify analytes on the basis of mass-to-charge (m/z) ratio. Although this technique has been used in research and specialized clinical laboratories for decades, however, in recent years, MS has been increasingly used in routine clinical laboratories. MS, especially when coupled to gas chromatography or liquid chromatography, provides very specific and often sensitive analysis of many analytes. Other advantages of MS include simultaneous analysis of multiple analytes (>100) and generally without need for specialized reagents. Commonly measured analytes by MS include drugs,...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry
We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine by LC-MS/MS. Method for Analyzing Isomers Without Chromatographic Separation
Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Presence of succinic acid (SA) complicates the analysis because mass spectra of MMA and SA are indistinguishable, when analyzed in negative ion mode and the peaks are difficult to resolve chromatographically. We developed a method for the selective analysis of MMA that exploits the significant difference in fragmentati...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of Metanephrine and Normetanephrine in Urine Using Liquid Chromatography-Tandem Mass Spectrometry
Measuring urinary metanephrines aides in the diagnosis of pheochromocytomas–catecholamine producing tumors. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows for greater sensitivity and simpler sample preparation as compared with other techniques. Here we describe a simple LC-MS/MS method for measuring metanephrines in urine. Each urine sample was treated with diphenylboronic acid to create boronate complexes, and then applied to a Bond-Elut Plexa cartridge. After solid phase extraction, samples were concentrated and analyzed on an Atlantis T3 column with chromatographic run time totaling 8.5 min. MS/M...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Free Metanephrines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry
We describe here a liquid chromatography-tandem mass spectrometry method to measure free metanephrines in plasma. Free metanephrine and normetanephrine are extracted via solid-phase extraction. After extraction and evaporation, the reconstituted supernatant is analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS is set to selective reaction monitoring mode (180.1 → 148.1 m/z for metanephrine, 183.1 → 168.1 for d3-metanephrine, 166.1 → 134.1 m/z for normetanephrine, and 169.1 → 137.2 ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Insulin-Like Growth Factor 1 in Serum by Liquid Chromatography High Resolution Accurate-Mass Mass Spectrometry
Insulin-like growth factor 1 (IGF-1) is a 70 amino acid peptide hormone which acts as the principal mediator of the effects of growth hormone (GH). Due to a wide variability in circulating concentration of GH, IGF-1 quantitation is the first step in the diagnosis of GH excess or deficiency. Majority (>95 %) of IGF-1 circulates as a ternary complex along with its principle binding protein insulin-like growth factor 1 binding protein 3 (IGFBP-3) and acid labile subunit. The assay design approach for IGF-1 quantitation has to include a step to dissociate IGF-1 from its ternary complex. Several commercial assays employ a bu...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Insulin Analogues in Serum Using Immunoaffinity Extraction, Liquid Chromatography, and Tandem Mass Spectrometry
We present an immunoaffinity extraction and LC-MS/MS method capable of independent identification and quantitation of native sequence insulins (endogenous, Insulin Regular, Insulin NPH), and analogues Glargine, Lispro, Detemir, and Aspart with an analytical sensitivity for endogenous insulin of between 1 and 2 μU/mL in patient serum samples. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry
We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directl...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Direct Measurement of Free Estradiol in Human Serum and Plasma by Equilibrium Dialysis-Liquid Chromatography-Tandem Mass Spectrometry
We describe a direct method of measurement of free estradiol using equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum aliquots and internal standards are extracted by liquid-liquid extraction using methyl-tert-butyl ether (MTBE) followed by derivatization with dansyl chloride. An API 5500 mass spectrometer operated in positive electrospray mode is used for detection. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Simultaneous Quantitation of Estradiol and Estrone in Serum Using Liquid Chromatography Mass Spectrometry
Accurate measurement of the endogenous estrogens, estrone (E1) and estradiol (E2), is important in the clinical diagnosis and monitoring of multiple disorders. Typically, given the efficacy and low cost, radioimmunoassays (RIA) and enzyme-linked immunoassays (EIA) are used to quantify these hormones in biological samples. Unfortunately, at low levels these assays lack the necessary sensitivity and specificity for diagnosis of certain disorders in adult and pediatric endocrinology and oncology. In response to this need, we developed a fast and sensitive high performance liquid chromatography negative electrospray ionization...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 56™ Mass Spectrometer
Triosephosphate isomerase (TPI) is a glycolytic enzyme which catalyzes the interconversion between glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). TPI deficiency results in accumulation of DHAP in human red blood cells and other tissues. The disease is characterized by congenital hemolytic anemia, and progressive neuromuscular dysfunction. The laboratory diagnosis is generally made by measurement of TPI activity in RBCs. Measurement of DHAP can be useful in further confirmation and follow-up of the disease. We developed HPLC/TOF-MS method for quantitation of DHAP in RBCs. The method involves simple ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

siRNA Nanoparticles for Ultra-Long Gene Silencing In Vivo
Small interfering RNA (siRNA)-mediated gene silencing has shown prominent therapeutic effects in treating various diseases. However, adequate delivery and persistent gene silencing remain challenging. A nanoparticle-based delivery system which assembled by layering siRNAs between protease degradable polypeptides to show ultra-long gene silencing effect in vivo is developed. Gold nanoparticle is used as a scaffold for its unique properties including uniform size, biocompatibility, ready synthesis, and easy functionalization. A simple layer-by-layer fabrication approach, based on the electrostatic interaction between positiv...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Whole-Body Scanning PCR, a Tool for the Visualization of the In Vivo Biodistribution Pattern of Endogenous and Exogenous Oligonucleotides in Rodents
Characterizing the in vivo biodistribution pattern and relative expression levels of oligonucleotide-based molecules such as mRNA, miRNA, siRNA, and anti-miRNAs in animal models, could be a helpful first-step in the successful development of therapeutic oligonucleotides. Here we describe a simple procedure called “Whole-Body Scanning PCR” (WBS-PCR), which combines the power of PCR with that of imaging. WBS-PCR relies on 384 well-defined extractions across a mouse whole-body section followed by a single dilution step which renders the lysates compatible with various qPCR-based assays. The in vivo biodistribution...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Imaging of Electrotransferred siRNA
SiRNA delivery to the cytoplasm can be obtained through the application of calibrated electric field pulses to a mixture of cells and oligonucleotides. To investigate the uptake pathway, time lapse confocal fluorescence microscopy provides a direct visualization of the transfer. SiRNA is electrophoretically drifted directly to the cytoplasm during the pulse. No post pulse transfer is observed. The uploaded siRNA then freely diffuse in the cytoplasm with no access to the nuclei. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Indium-Labeling of siRNA for Small Animal SPECT Imaging
Ever since the discovery of RNA interference (RNAi), therapeutic delivery of siRNA has attracted a lot of interest. However, due to the nature and structure of siRNA, a carrier is needed for any mode of systemic treatment. Furthermore, specific imaging techniques are required to trace where the deposition of the siRNA occurs throughout the body after treatment. Tracking in vivo siRNA biodistribution allows understanding and interpreting therapeutics effects and side effects. A great advantage of noninvasive imaging techniques such as SPECT imaging is that several time points can be assessed in the same subject. Thus, the t...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Analyses of Tumor Burden In Vivo and Metastasis Ex Vivo Using Luciferase-Expressing Cancer Cells in an Orthotopic Mouse Model of Neuroblastoma
Cancer xenograft mouse models are useful for examining and understanding tumor growth and cancer progression in vivo. With the development of bioluminescent imaging, these parameters can now be monitored noninvasively with relative ease. Herein we describe imaging of luciferase-expressing cancer cells to quantitatively measure tumor burden in vivo and metastases ex vivo. Specifically, we detail the methodology to examine the effect of shRNA-mediated knockdown of a target gene on the growth and spread of neuroblastoma tumors in immune-deficient mice. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Targeted Delivery with Imaging Assessment of siRNA Expressing Nanocassettes into Cancer
Molecular therapy using small interfering RNA (siRNA) shows great promise in the development of novel therapeutics for cancer. Although various approaches have been developed for in vivo delivery of siRNAs into tumors, stability of siRNA in blood circulation, and low efficiency of siRNA delivery into tumor cells are the major obstacles for further translation into cancer therapeutics. In this protocol, we describe methods of the production of shRNA expressing DNA nanocassettes by PCR amplification of double-stranded DNA fragments containing a U6 promoter and a shRNA gene. Those DNA nanocassettes can be conjugated to the po...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Magnetic Resonance Spectroscopy of siRNA-Based Cancer Therapy
Small interfering RNA (siRNA) is routinely used as a biological tool to silence specific genes, and is under active investigation in cancer treatment strategies. Noninvasive magnetic resonance spectroscopy (MRS) provides the ability to assess the functional effects of siRNA-mediated gene silencing in cultured cancer cells, and following nanoparticle-based delivery in tumors in vivo. Here we describe the use of siRNA to downregulate choline kinase, a critical enzyme in choline phospholipid metabolism of cancer cells and tumors, and the use of 1H MRS of cells and 1H magnetic resonance spectroscopic imaging (MRSI) of tumors t...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

In Vivo Magnetic Resonance Imaging of Small Interfering RNA Nanodelivery to Pancreatic Islets
Pancreatic islet transplantation is a promising therapeutic approach for type 1 diabetes. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Imaging Functional Nucleic Acid Delivery to Skin
Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A ...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

MicroRNA Imaging in Combination with Diagnostic Ultrasound and Bubble Liposomes for MicroRNA Delivery
MicroRNA (miRNA) is expected to play an important role in the diagnosis and therapy of various diseases. In miRNA therapy, the development of delivery tools to the target site is considered to be essential. By using a delivery tool possessing imaging ability, miRNA colocalized with the carrier could be visualized after administration. We prepared polyethylene glycol (PEG)-modified liposomes containing echo-contrast gas, “Bubble liposomes” (BLs), and confirmed that BLs containing cationic lipid were capable of loading miRNA. Furthermore, we also achieved the imaging and delivery of systemically injected miRNA to...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILES
MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, ...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Microvesicles: Isolation, Characterization for In Vitro and In Vivo Procedures
Microvesicles and exosomes are released to the extracellular milieu and are detectable in body fluids. They act as unique vehicles for cargo transfer to other cells/tissues. They contain a set of membrane receptors, intracellular proteins and nucleic acids. Here, we describe basic techniques for their isolation and characterization from cell culture media or body fluids. We also describe critical techniques for characterizing their cargo (miRNAs) and their protein content. Finally, we present labeling methods for their use in in vitro procedures and for their in vivo delivery and subsequent analysis of their cargo transfer...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Controlling RNA Expression in Cancer Using Iron Oxide Nanoparticles Detectable by MRI and In Vivo Optical Imaging
Herein, we describe a protocol for the preparation of iron oxide nanoparticle-based contrast agents and drug delivery vehicles for noninvasive cancer imaging and therapy. In the first part of the chapter we describe the details of the contrast agent synthesis, functionalization, and characterization. In the second part we describe the methods for tumor imaging using the synthesized particles with noninvasive T2-weighted magnetic resonance imaging (MRI) and in vivo near infrared optical imaging. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Hypoxia-Responsive Copolymer for siRNA Delivery
A wide variety of nanomedicine has been designed for cancer therapy. Herein, we describe the synthesis and evaluation of a hypoxia-responsive copolymer for siRNA delivery (Perche et al., Angew Chem Int Ed Engl 53:3362–3366, 2014). The synthesis is achieved using established coupling chemistry and accessible purification procedures. A polyelectrolyte-lipid conjugate (polyethyleneimine 1.8 kDa-dioleyl-phosphatidylinositol, PEI-PE) and polyethylene glycol 2000 (PEG) were assembled via the hypoxia-sensitive azobenzene (Azo) unit to obtain the PEG-Azo-PEI-DOPE copolymer. This copolymer can condense siRNA and shows hypoxia...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Molecular Beacon-Based MicroRNA Imaging During Neurogenesis
The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quen...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Sensing miRNA: Signal Amplification by Cognate RISC for Intracellular Detection of miRNA in Live Cells
The ability to detect miRNA expression in live cells would leave these cells available for further manipulation or culture. Here, we describe the design of a miRNA sensor oligonucleotide whose sequence mimics the target mRNA. The sensor has a fluorescent label on one end of the oligo and a quencher on the other. When inside the cell, the sensor is recognized by its cognate miRNA-RISC and gets cleaved, setting the fluorophore free from its quencher. This results in fluorescence “turn on.” Since cleavage by the RISC complex is an enzymatic process, the described approach has a very high level of sensitivity (nM)....
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Optogenetics: Optical Control of a Photoactivatable Rac in Living Cells
Recent developments in optogenetics have extended optical control of signaling to intracellular proteins, including Rac, a small G protein in the Rho family. A blue light-sensing LOV (light, oxygen, or voltage) domain derived from Avena sativa (oat) phototropin was fused to the N-terminus of a constitutively active mutant of Rac, via an α-helix (Jα) that is conserved among plant phototropins. The fused LOV domain occluded binding of downstream effectors to Rac in the dark. Exposure to blue light caused a conformational change of the LOV domain and unwinding of the Jα helix, relieving steric inhibition. Th...
Source: Springer protocols feed by Imaging/Radiology - November 13, 2014 Category: Radiology Source Type: news

Direct Stochastic Optical Reconstruction Microscopy (dSTORM)
Single-molecule localization-based super-resolution microscopy can be performed with regular, bright, and photostable organic fluorophores. We review a concept termed direct stochastic optical reconstruction microscopy (dSTORM), which operates conventional fluorophores as photoswitches and provides an optical resolution of ~20 nm. We introduce the principle of dSTORM, illustrate experimental schemes, and discuss approaches for data analysis. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 13, 2014 Category: Radiology Source Type: news

Three-Dimensional Photoactivated Localization Microscopy with Genetically Expressed Probes
We describe methods for converting a 2D PALM into a system capable of acquiring such 3D images, with a particular emphasis on instrumentation that is compatible with choosing relatively dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs) as PALM probes. After reviewing the basics of 2D PALM, we detail astigmatic and multiphoton imaging approaches well suited to working with PA-FPs. We also discuss the use of open-source localization software appropriate for 3D PALM. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 13, 2014 Category: Radiology Source Type: news

Application of STED Microscopy to Cell Biology Questions
The increasing interest in “seeing” the molecular environment in biological systems has led to the recent quest for breaking the diffraction barrier in far-field fluorescence microscopy. The first nanoscopy method successfully applied to conventional biological probes was stimulated emission depletion microscopy (STED). It is based on a physical principle that instantly delivers diffraction-unlimited images, with no need for further computational processing: the excitation laser beam is overlaid with a doughnut-shaped depleting beam that switches off previously excited fluorophores, thereby resulting in what is...
Source: Springer protocols feed by Imaging/Radiology - November 13, 2014 Category: Radiology Source Type: news

4Pi Microscopy of the Nuclear Pore Complex
4Pi microscopy is a far-field fluorescence microscopy technique, in which the wave fronts of two opposing illuminating beams are adjusted to constructively interfere in a common focus. This yields a diffraction pattern in the direction of the optical axis, which essentially consists of a main focal spot accompanied by two smaller side lobes. At optimal conditions, the main peak of this so-called point spread function has a full width at half maximum: fixed phrase of 100 nm in the direction of the optical axis, and thus is 6–7-fold smaller than that of a confocal microscope. In this chapter, we describe the basic feat...
Source: Springer protocols feed by Imaging/Radiology - November 13, 2014 Category: Radiology Source Type: news

Practical Structured Illumination Microscopy
Structured illumination microscopy (SIM) is a method that can double the spatial resolution of wide-field fluorescence microscopy in three dimensions by using spatially structured illumination light. In this chapter, we introduce the basic principles of SIM and describe in detail several different implementations based on either a diffraction grating or liquid crystal spatial light modulators. We also describe nonlinear SIM, a method that in theory can achieve unlimited resolution. In addition, we discuss a number of key points important for high-resolution imaging. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 13, 2014 Category: Radiology Source Type: news