In the present study, sample labelling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, Inc.). Briefly, total RNA from each sample was linearly amplified and labelled with Cy3-UTP (Enzo Life Sciences, Inc.). The labelled cRNAs were purified using an RNeasy Mini kit (Qiagen, Inc.). The concentration and specific activity of the labelled cRNAs (pmol Cy3/µg cRNA) were measured using a spectrophotometer. A total of 1 µg of each labelled cRNA was fragmented by the addition of 11 µl 10X blocking agent (LMAI Bio) and 2.2 µl 25X fragmentation buffer (Agilent Technologies, Inc.), then heated at 60°C for 30 min. Subsequently, 55 µl 2X GEx Hybridization Buffer HI-RPM (Agilent Technologies, Inc.) were added to dilute the labelled cRNA and 100 µl hybridization solution (Agilent Technologies, Inc.) were dispensed into the gasket slide and assembled on the gene expression microarray slide. The slides were incubated for 17 h at 65°C in the aforementioned Agilent hybridization oven. The hybridized arrays were washed, fixed and scanned using the Agilent DNA Microarray Scanner (cat. no. G2505C; Agilent Technologies, Inc.).
Hybridization solution
Manufactured by Agilent Technologies
Sourced in United States
Hybridization solution is a laboratory reagent used in the process of hybridization, which is a fundamental technique in molecular biology and genetics. The solution facilitates the formation of stable bonds between complementary nucleic acid sequences, such as between a target DNA or RNA and a labeled probe. This process allows for the detection and analysis of specific genetic targets within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Gasket 8 plex slide, by Agilent Technologies (5 mentions)
Low input quick amp labeling kit, by Agilent Technologies (4 mentions)
Agilent sureprint g3 human ge 8 60 k array, by Agilent Technologies (3 mentions)
Genespringgx 7, by Agilent Technologies (2 mentions)
Hybridization solution, by Molecular Research Center (2 mentions)
Hybridization tube, by Agilent Technologies (2 mentions)
Prime it 2 random primer labeling kit, by Agilent Technologies (2 mentions)
Sequagel ureagel system, by National Diagnostics (2 mentions)
Ssc solution, by Thermo Fisher Scientific (2 mentions)
Qiazol, by Qiagen (2 mentions)
Fragmentation buffer, by Agilent Technologies (1 mentions)
Cy3 utp, by Enzo Life Sciences (1 mentions)
Agilent one color microarray based gene expression analysis protocol, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Microarray scanner, by Agilent Technologies (1 mentions)
Low input quick amp labelling kit, by Agilent Technologies (1 mentions)
Gene expression wash buffer 1, by Agilent Technologies (1 mentions)
Gene expression wash buffer 2, by Agilent Technologies (1 mentions)
Sureprint g3 human ge 8 60 k array, by Agilent Technologies (1 mentions)
Feature extraction version 10, by Agilent Technologies (1 mentions)
9 protocols using hybridization solution
1
Microarray Gene Expression Analysis Protocol
2
Transcriptomic Analysis of MCF7 Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs (50 ng) from MCF7-luc derivatives (MCF7-luc anti-NC, MCF7-luc anti-miR-27b, MCF7-luc miR-27b o.e. and MCF7-ENPP1-MF cells) were labelled and amplified using the Low Input Quick Amp labelling kit (Agilent Technologies). The Cy3-labelled RNAs were resuspended in 40 μl of hybridization solution (Agilent Technologies), applied to a SurePrint G3 Human GE 8 × 60 K array (Agilent Technologies) and covered with a Gasket 8-plex slide (Agilent Technologies). The slides were hybridized for 17 h at 65 °C, washed with Gene Expression Wash Buffer 1 (Agilent Technologies) for 1 min at room temperature, washed with Gene Expression Wash Buffer 2 (Agilent Technologies) for 1 min at 37 °C and then air-dried. The arrays from three independent experiments were analysed using microarray scanner (Agilent Technologies). Gene expression levels were calculated using Feature Extraction version 10.7.3.1 (Agilent Technologies). The normalized and log-transformed intensity values were then analysed using GeneSpring GX 7.3.1 (Agilent Technologies).
3
Transcriptome Analysis of Labeled RNA Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each total RNA sample (200 ng) was labeled and amplified using a Low Input Quick Amp Labeling Kit (Agilent Technologies, CA). The Cy3-labeled aRNAs were resuspended in 50 μL of hybridization solution (Agilent Technologies, CA). After the labeled aRNAs were placed on an Agilent SurePrint G3 Human GE 8 × 60 K array (Agilent Technologies, CA) and covered by a Gasket 8-plex slide (Agilent technologies, CA), the slides were hybridized for 17 hours in a 65°C oven. The hybridized slides were washed in 2 × saline-sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) for 2 min, 1 × SSC for 3 min, and then 0.2 × SSC for 2 min at room temperature. The slides were centrifuged at 3000 rpm for 20 sec to dry.
4
Gene Expression Profiling of Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHOK‐KGM, IHOK‐EF, and IHOK‐EFKGM were cultured to 70% confluency. Total RNA was isolated using RNeasy kit (Qiagen) according to the manufacturer's instructions. Each total RNA sample (200 ng) was labeled and amplified using Low Input Quick Amp labeling kit (Agilent technologies, CA). The Cy3‐labeled aRNAs were resuspended in 50 µl of hybridization solution (Agilent technologies). After labeled aRNAs were placed on Agilent Sure Print G3 Human GE 8 × 60K array (Agilent technologies) and covered by a Gasket 8‐plex slide (Agilent technologies), the arrays were analyzed using an Agilent scanner with associated software. Gene expression levels were calculated with Feature Extraction v10.7.3.1 (Agilent technologies). Relative signal intensity for each gene was generated using the Robust Multi‐Array Average algorithm. The data were processed based on the median polish normalization method using Gene Spring GX 7.3.1 (Agilent technologies). The normalized and log‐transformed intensity values were then analyzed using Gene Spring GX 7.3.1 (Agilent technologies).
5
Northern Blot Analysis of SNORA31
6
GLP-1 Secretion Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were carried out in similar GLP-1 secretion study processes and we used 100 μg/mL of the HFBF. Three days before the experiments, the cells were seeded at 1 × 107 cells/well into 6-well culture plates precoated with Matrigel (BD Biosciences, USA). Other procedures were performed in the same way as described in the GLP-1 assay. Total RNA was extracted by using Hybrid-RTM (GeneAll Biotechnology, Korea) according to the manufacturer's instructions. The microarray of each total RNA sample (200 ng) was labeled and amplified using a Low Input Quick Amp Labeling Kit (Agilent Technologies, USA) [10 ], and the Cy3-labeled aRNAs were resuspended in 50 μL of hybridization solution (Agilent Technologies, USA). After the aRNAs were placed on an Agilent SurePrint G3 Human GE 8 × 60 K Array (Agilent Technologies, USA) and covered by a Gasket 8-plex slide (Agilent Technologies, USA), the slides were hybridized for 17 hours in a 65°C oven. The hybridized slides were washed in 2 × saline-sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) for 2 min, 1 × SSC for 3 min, and then 0.2 × SSC for 2 min at room temperature. The slides were centrifuged at 3000 rpm for 20 sec to dry.
7
Northern Blot Analysis of SNORA31
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted in Qiazol (Qiagen, #79306). Acrylamide gels were prepared with the SequaGel UreaGel System (National Diagnostics), according to the manufacturer’s protocol. Equal amounts of RNA sample and RNA loading dye (BioLabs, #B0363A) were mixed and heated at 70°C for 5 minutes to denature the RNA. After migration in the gel, the RNA was transferred to a nylon membrane (GE Healthcare, #RPN303B) and fixed with an EDC solution for cross-linking. The gel was stained with ethidium bromide for a few minutes to visualize the ribosomal RNA bands. The immobilized RNA membrane was then wetted with SSC solution (Invitrogen, #15557–044) placed in a hybridization tube with hybridization solution (Molecular Research Center, #HS114F), and heated for 3 hours at 42°C. The 32P-labeled double-stranded full-length SNORA31 probe, labeled with the Prime-It II Random Primer Labeling Kit (Agilent Technologies, #300385), was heated at 100°C for 10 minutes, added to the hybridization solution and the hybridization tube was then incubated overnight at 42°C. The membrane was washed several times with SSC solutions of increasing stringency, and the radioactive signal was detected by placing the membrane against photographic film.
8
Microarray Analysis of Liver lncRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from homogenized liver tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA labeled with a fluorescent dye (cyanine 3-dCTP) was produced by Eberwine's linear RNA amplification method and subsequent enzymatic reaction. Double-stranded cDNAs (containing the T7 RNA polymerase promoter sequence) were synthesized from 1 µg total RNA using the CbcScript reverse transcriptase with cDNA synthesis system according to the manufacturer's protocol (CapitalBio Technology, Inc.) with the T7 Oligo(dT) and T7 Oligo(dN). After completing double-stranded cDNA (dsDNA) synthesis using DNA polymerase and RNase H (MACHEREY-NAGEL GmbH & Co. KG), the dsDNA products were purified using a PCR NucleoSpin Extract II Kit (MACHEREY-NAGEL GmbH & Co. KG). 14 µl DNA was denatured in hybridization solution (Agilent Technologies, Inc.) at 95˚C for 3 min prior to loading onto a microarray (Mouse lncRNA Array v1.0, 4x180K; CapitalBio Technology, Inc.). Arrays were hybridized in a hybridization oven (Agilent Technologies, Inc.) overnight at a rotation speed of 20 rpm and a temperature of 42˚C and washed with two consecutive solutions (0.2% SDS, 2X SSC at 42˚C for 5 min and 0.2X SSC at 23-26˚C for 5 min).
9
Rat Gene Expression Microarray Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each total RNA sample (100 ng) was labeled and amplified using Low Input Quick Amp labeling kit (Agilent Technologies, CA, USA). The Cy3-labeled aRNAs were resuspended in 50 µl of hybridization solution (Agilent Technologies, Santa Clara, CA, USA). After labeled aRNAs were placed on Agilent SurePrint G3 Rat GE 8X60K array (Agilent Technologies) and covered by a Gasket 8-plex slide (Agilent Technologies). The slides were hybridized for 17 hours at 65℃ oven. The hybridized slides were washed in 2× saline sodium citrate (SSC), 0.1% sodium dodecyl sulfate for 2 minutes, 1× SSC for 3 minutes, and then 0.2× SSC for 2 minutes at room temperature. The slides were centrifuged at 3,000 rpm for 20 seconds to dry.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!