Isolation and quality control of RNA Lysis and homogeneization is performed with 1 mL pre-warmed (60°C) TRIzol Reagent (Invitrogen Cat. No. 15596-026) and 5 mm stainless steel beads (Qiagen Cat. No. 69989, 2 times 2 min at 25 Hz in tissue lyser Mixer Mill 300 for 2-3 mm Ø biopsies). The extraction is completed by adding Phase Lock Gel (Eppendorf Cat. No. 0032007.970) during the phase extraction. Molecular biology grade glycogen (Roche Diagnostics GmbH, Cat. N. 901393) is added to the aqueous phase during the precipitation step. After purification step on RNeasy Microkit columns (Qiagen, Cat. No. 74004), the final cleaned total RNA is eluted in 14 µL of RNase free water and stored at -80°C. RNA levels, quality and purity are assessed with the use of RNA 6000 Nano and Pico assay on the Agilent 2100 Bioanalyzer. Hybridization A Tecan HS 4800 Hybridization station from Tecan, Inc. is used for prewash/hybridization/postwash of the microarrays. Prewash consists of 3 cycles with wash buffer at RT/75°C/50°C (each 20 s) followed by an additional 10 s wash with hybridization buffer at 50°C. One hundred L sample (100 ng labeled RNA) in hybridization buffer is then injected into the flow chambers at 50°C and agitated for 1 min. Subsequently, after 10 min at 75°C, the temperature is adjusted to 42°C for 16 h (agitation). The postwash consists in 4 cycles wash buffer at 42°C followed by an additional wash at 23°C (each 20 s). Finally, the slides are washed 3 times with diluted wash buffer at RT, dried in N2 stream, and scanned immediately with an Agilent LS fluorescence scanner (gain 80%). Analysis of fluorescence images The fluorescence images is analyzed by means of Array Pro (Mediacybernetics, Inc. US). Net signals is calculated by subtracting the 75 percentile of local corner background from trimmed mean of each spot (20% trimming on both sides of the intensity histogram). Individual samples are processed on separate slides in single color mode (Cy5). The 75 percentile of intensity histogram is adjusted to 200 counts (a.u.) for normalization of the microarrays. The image analysis is configured in a way that allowed an automated assessment of the quality of individual fluorescence spots (criteria: local fluorescence background, spots symmetry, presence in "salt image"/Print QC). Only microarrays with >98% qualified spots are used for further analysis.
Support
glass
Description
Novachip is an evanescent resonator microarray platform. The approach takes advantage of a phenomenon that has been attributed as abnormal reflection. Excitation photons incident on the chip under resonance conditions couple into a thin corrugated metal oxide surface at the site of incidence. As a result of the transducer geometry, the energy is locally confined into the thin corrugated layer of high refractive index material. Consequently, strong electromagnetic fields are generated at the surface of the chip. The effect has been attributed as evanescent resonance and leads to increased fluorescence intensity of chromophores close to the surface. The effective field strength can be increased up to 100-fold by the confinement of the available excitation energy, depending on the optical properties of the used optical detection system. The Novachips used for the experiments described in the present paper have a resonance angle of 2° with respect to normal for TE polarized light of 633 nm wavelength. The observed increase of fluorescence intensity with the used Agilent microarray laser scanner was about 5-fold compared to glass slides processed under identical conditions. The slides were cleaned and the AROS human v3 oligonucleotide set from Operon Inc. was arrayed with a Microgrid printer at 23°C and 55-60% relative humidity (total approx. 37K features, print buffer 4x SSC / 0.001% Sarcosyl). Scatter light scans (Tecan Laser Scanner) were used to assess the quality of the printed slides ("salt images"). Slides were stored sealed until use.
N-ACETYLGLUCOSAMINYL-PHOSPHATIDYLINOSITOL BIOSYNTHETIC PROTEIN (GLCNAC-PI SYNTHESIS PROTEIN) (PHOSPHATIDYLINOSITOL-GLYCAN BIOSYNTHESIS, CLASS A PROTEIN) (PIG-A)
Less...
More...