Agilent wash buffer kit

Human 4 × 180 K lncRNA arrays contained 37581 lncRNAs and 34235 mRNAs. Total RNA was obtained as described above. Approximately 200 ng of total RNA from each sample was used for the microarray analyses. Briefly, followed by spiking with an RNA Spike-In Kit with one color (Agilent), total RNA was reversely transcribed into cDNA and then converted into Cyanine-3 labeled cRNA. Cyanine-3-labeled cRNA sample was fragmented and then hybridized at 65°C for 17 h using an Agilent Gene Expression Hybridization Kit in hybridization chamber gasket slides (Agilent). After hybridization, the microarrays were washed with an Agilent Wash Buffer kit (Agilent) and scanned with an Agilent microarray scanner. The resulting images were analyzed using Agilent's Feature Extraction software v10.7 and Agilent GeneSpring.

For each sample,
a predetermined amount of synthetic DNA was added to 1 μg of
extracted DNA as a spike-in control. Subsequently, the mixed DNA was
labeled with Cy-3 (or Cy-5) fluorescent dye (GE Healthcare, Vacaville,
CA, USA) using random primers and Klenow fragment of DNA polymerase
I. Labeled DNA was then purified using a QIAquick Purification kit
(Qiagen, Valencia, CA, USA), and the NanoDrop 8000 UV–vis Spectrophotometer
(Thermo Scientific; Waltham, MA) was used to measure the yield and
degree of labeling. Each sample was supplemented with a total of 42 μL
of buffer containing 1× HI-RPM hybridization buffer, 1×
aCGH blocking agent, 0.05 μg/μL of Cot-1 DNA, and 10%
formamide. The mixture was then vortexed thoroughly, spun down, and
incubated at 95 °C for 3 min, followed by incubation at 37 °C
for 30 min. The samples were subsequently hybridized with CyanoStrainChip
at 67 °C for 24 h with a rotation at 20 rpm in an Agilent hybridization
oven (Agilent Technologies, Inc., Santa Clara, CA, USA). For posthybridization
washing, an Agilent Wash Buffer Kit (Agilent, Santa Clara, CA) was
used for removing nonhybridized or partially hybridized labeled sample
DNA from the array’s surface to minimize signal noise.

Total RNA (200 ng each) from these tissue samples was reversely transcribed into cDNA using an RNA Spike In Kit with one-color (Agilent) in the presence of 0.8 µL of Random Primer and 2 µL of Spike Mix. These cDNA samples were then cleaned and labeled in accordance with the Agilent Gene Expression Analysis protocol using Low Input Quick-Amp Labeling Kit, one-color (Agilent). These labeled cDNA samples were used as probes to hybridize to microarrays for 17 h at 65°C using an Agilent Gene Expression Hybridization Kit in hybridization chamber gasket slides (Agilent). After hybridization, the microarrays were washed with an Agilent Wash Buffer kit (Agilent) and scanned with an Agilent microarray scanner.