Agilent Manuals

Application Note Abstract Microarray-based gene expression hybridization is a powerful and proven technique for studying differential gene expression signatures in cancer. RNA extracted from freshly frozen tissues is optimal for microarray analysis; however, in many cases, formalin-fixed paraffi n-embedded (FFPE) tissues are the only samples available. To work with these diffi cult samples, Agilent has developed an FFPE sample protocol optimized for use with Agilent gene expression microarrays. To test the effectiveness of this protocol, total RNA was extracted from a quadruplicate set of colon tumor (adenocarcinoma), and matched normal colon FFPE and fresh frozen tissues. RNA purity and quality were determined, and the RNA was amplifi ed using a system in which a reverse transcription-generated cDNA library is subjected to limited PCR amplifi cation. The resulting cDNA target was chemically labeled. Analysis of the microarray results revealed a reduced sensitivity of detection for the FFPE-derived RNA samples; however, the lists of detected genes showed high overlap between the FFPE and frozen samples. Comparison of gene expression signals demonstrated excellent assay reproducibility between the technical repli- cates. Principal component analysis of array signals demonstrated clear separa- tion between the tumor and normal samples with grouping of replicate samples. Statistical analysis of the expression level variations between tumor and normal samples revealed that both the FFPE and frozen samples showed similar expression level changes.

This document describes a one-color gene expression capability developed by Agilent Technologies, as well as the performance and advantages of this system.

This Application Note describes a high-speed, high-resolution capillary electrophoresis method of heparin and related impurities using highly concentrated buffer in a 25 µm bubble cell capillary. The method can be used to detect heparin and related impurities in a short time.

This Application Note describes the rapid separation of metabolites generated from in-vitro experiments using the Agilent 1290 Infinity LC system. It also explains the fast acquisition of TOF mass spectra using Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight LC/MS systems. The software-assisted identification of main metabolites from in-vitro experiments using Agilent MassHunter Metabolite Identification software is discussed. The generation of reports for the identified metabolites using Agilent MassHunter software is also covered.

This Application Note demonstrates: The use of the Agilent 1200 Rapid Resolution LC (RRLC) system for high resolution separation of metabolites from an in-vitro metabolism experiment. The use of the Agilent 6520 QTOF mass spectrometer for the acquisition of data for computer assisted metabolite identification. The use of the Agilent MassHunter Metabolite identification software for highly productive identification of expected metabolites. The results of the Metabolite ID data analysis for expected metabolites of the pharmaceutical drug nefazodone

This Application Note demonstrates: (1) The use of the Agilent 1200 Rapid Resolution LC (RRLC) system for high resolution separation of metabolites from an in-vitro metabolism experiment. (2) The use of the Agilent 6520 QTOF mass spectrometer for the acquisition of data for computer assisted metabolite identification. (3) The use of the Agilent MassHunter Metabolite identification software for highly productive identification of expected metabolites. (4) The results of the Metabolite ID data analysis for expected metabolites of the pharmaceutical drug nefazodone.

This Application Note evaluates the performance of the Agilent 1290 Infinity LC system for the determination of impurities and related substances of metoclopramide hydrochloride in a pharmaceutical formulation. The translation of a conventional liquid chromatography (LC) method on a 1200 Series HPLC system to an ultra high pressure method on an Agilent 1290 Infinity LC system is discussed. Method translation is relatively easy and temperature fine-tuning is the most important parameter to obtain the same selectivity for the different impurities. Pressures as high as 1070 bar were applied during method development. The final high productivity method is carried out at 880 bar in an analysis time of 3.5 min which is approximately 4 times faster than the original HPLC method but with the same accuracy. A validation study was carried out to demonstrate the performance of the Agilent 1290 Infinity LC system. Limits of detection for the impurities were as low as 0.001 % w/w relative to the main compound using the new diode array detector (DAD). This is more than one order of magnitude lower than required.

This Application Note describes the use of highly sulphonated cyclodextrins for the analysis of chiral analytes. By using small 25 µm id capillaries with an extended pathlength of 125 µm id the Joule heating can be controlled while enhancing sensitivity.

This document introduces a feasibility study of a capillary electrophoresis-mass spectrometry (CE-MS) technique for toxicological drug screening in blood samples. The study shows that the Agilent LC/MSD Trap system can detect drugs at levels as low as 20 ng/mL in whole blood, suggesting that it may be a suitable tool for screening whole blood samples for drugs. Additionally, the system's capability of generating MSn data allows reliable identification of detected drugs, suggesting that both screening and identification of drugs can be accomplished in one or two injections using the same instrument system.

This Application Note describes methods of using the Agilent Capillary Electrophoresis (CE) system to separate single- and double-stranded DNA, single-stranded RNA and antisense oligonucleotides. Oligonucleotides with a chain length of 5 to 80 nucleotides are separated using polymer solution A and polymer solution B. The application note describes how to achieve high reproducibility in separations of oligonucleotides.

This document describes the method and results of analyzing anions in power plant waters using capillary electrophoresis. Anion analysis is performed using indirect UV detection, allowing for fast and reliable analysis of low ppb levels of small inorganic and organic anions.

This application note describes a method for the analysis of aromatic amines in leather extracts using CE-MS. This method provides specific identification of analytes in complex matrices that would present difficulties for HPLC. Organic extracts of leather fit this description exactly, therefore CE-MS is the method of choice.

This document describes the use of capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS) to analyze hallucinogenic mushroom extracts. The data presented here demonstrate feasibility. Additional method development and/or validation may be required for routine use.