Total RNA was prepared from mammary tumors and from matched, adjacent, normal transgenic mammary glands. Exiqon microarrays (Qiagen Inc, Germantown, MD) were used to assess differential miRNA expression. Four independent sets of tumors and their matched, normal transgenic mammary glands were compared. Three sets were assayed in duplicate (dye swap) and one set was tested in only a single assay. The dye swap assay was not performed for this pair. Total RNA (1000 ng) was labeled using either Hy5 or Hy3 dye following the manufacturer’s protocol for the miRCURY LNA microRNA Array (Exiqon/Qiagen). The Hy5-labeled samples and Hy3-labeled samples were mixed pairwise and hybridized to the array. Hybridization and wash steps were performed using a Tecan HS400 Hybridization Station (Tecan, Austria). The miRCURY LNA array slides were scanned using GenePix 4200 (Molecular Devices, San Jose, CA). Differential miRNA expression was validated by reverse-transcriptase quantitative PCR (RT-qPCR). RT-qPCR was performed utilizing SYBR assays obtained from Exiqon (Qiagen Inc.), using the same four pairs of tumors and matched tissues that were used for the microarray experiments. These experiments also included two additional tumor-matched tissue pairs. Expression of potential downstream target genes was assessed using RT-qPCR.
Genepix 4200
Manufactured by Molecular Devices
Sourced in Uruguay
The GenePix 4200 is a high-performance microarray scanner designed for gene expression analysis. It features a dual-laser system that can simultaneously excite and detect fluorescent signals from multiple dyes. The scanner offers a range of scanning resolutions and provides rapid data acquisition capabilities.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using genepix 4200
1
Profiling Mammary Tumor miRNA Expression
2
Microarray-based Single Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Epoxy-functionalized
glass slides (Super
epoxy 2, Arrayit Corporation) were coated with 100 μg/mL ovalbumin
in PBS overnight at 4 °C and then rinsed with PBS and blocked
with 1 mg/mL BSA/PBS for 30 min. After incubation of the cell-loaded
array, the ovalbumin-coated glass slide was then placed onto the loaded
array for printing. The microarray and glass slide were held together
by compression in a hybridization chamber (Agilent Technologies, G2534A)
and incubated for 2 h at 37 °C with 5% CO2 as described
previously.6 (link) The glass slide was then separated
from the array and placed in PBS. After microengraving, slides were
incubated for 30 min with blocking buffer (PBS, 3% w/v milk powder
and 0.05% (v/v) Tween-20), washed with PBST (PBS + 0.05% v/v Tween-20),
and then incubated with fluorescent antibodies (Alexa Fluor 488 goat
anti-mouse IgG) at 2 μg/mL for 45 min at 25 °C. The slides
were washed with PBST and PBS, rinsed briefly with water, and dried
with a N2 stream. Slides were scanned using a Genepix 4200AL
microarray scanner (Molecular Devices). The median fluorescence intensity
of each element was analyzed using Genepix Pro. Data extracted from
both on-chip cytometry and printed proteins were matched in Microsoft
Excel using unique identifiers assigned to each well within the array.
The dataset was filtered to include wells containing only single cells.
glass slides (Super
epoxy 2, Arrayit Corporation) were coated with 100 μg/mL ovalbumin
in PBS overnight at 4 °C and then rinsed with PBS and blocked
with 1 mg/mL BSA/PBS for 30 min. After incubation of the cell-loaded
array, the ovalbumin-coated glass slide was then placed onto the loaded
array for printing. The microarray and glass slide were held together
by compression in a hybridization chamber (Agilent Technologies, G2534A)
and incubated for 2 h at 37 °C with 5% CO2 as described
previously.6 (link) The glass slide was then separated
from the array and placed in PBS. After microengraving, slides were
incubated for 30 min with blocking buffer (PBS, 3% w/v milk powder
and 0.05% (v/v) Tween-20), washed with PBST (PBS + 0.05% v/v Tween-20),
and then incubated with fluorescent antibodies (Alexa Fluor 488 goat
anti-mouse IgG) at 2 μg/mL for 45 min at 25 °C. The slides
were washed with PBST and PBS, rinsed briefly with water, and dried
with a N2 stream. Slides were scanned using a Genepix 4200AL
microarray scanner (Molecular Devices). The median fluorescence intensity
of each element was analyzed using Genepix Pro. Data extracted from
both on-chip cytometry and printed proteins were matched in Microsoft
Excel using unique identifiers assigned to each well within the array.
The dataset was filtered to include wells containing only single cells.
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