Probe labelling and hybridization were performed with Roche NimbleGen kit using the standard protocol for eukaryotic RNA samples and NimbleGen mouse gene expression 12x135K array (Roche, cat. no. 05543797001). The cDNA samples were labelled with Cy3 using a NimbleGen One-Color DNA labelling kit (Roche, cat. no. 06370411001) and hybridized to slides using a NimbleGen hybridization system (Roche, cat. no. 05583683001). The slides were scanned using MS200 Scanner (Roche, NimbleGen) at 2 μm resolution by using high-sensitivity and autogain settings.
Nimblegen hybridization system
Manufactured by Roche
Sourced in Germany, United States
The NimbleGen Hybridization System is a laboratory equipment designed for performing microarray hybridization. It facilitates the hybridization process, which is a crucial step in microarray analysis. The system provides a controlled environment to ensure the accurate and efficient binding of labeled samples to the microarray probes.
Automatically generated - may contain errors
Lab products found in correlation
Nimblegen one color dna labeling kit, by Roche (5 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Axon genepix 4000b microarray scanner, by Molecular Devices (2 mentions)
Nimblegen ms 200 microarray scanner, by Roche (2 mentions)
Nimblegen one color dna labelling kit, by Roche (1 mentions)
Nimblegen mouse gene expression 12x135k array, by Roche (1 mentions)
Ms200 scanner, by Roche (1 mentions)
Nimblescan software, by Roche (1 mentions)
Nimblegen wash buffer kit, by Roche (1 mentions)
Arraycontrol rna spikes, by Thermo Fisher Scientific (1 mentions)
Agilent dna microarray scanner g2565ca, by Agilent Technologies (1 mentions)
Elx800 absorbance microplate reader, by Agilent Technologies (1 mentions)
Agilent genespring software, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Nimblescan, by Roche (1 mentions)
Nimblegen 12 135 k array, by Roche (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
7 protocols using nimblegen hybridization system
1
Probe Labeling and Hybridization for Mouse Gene Expression
2
Long Non-Coding RNA Expression Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A Human lncRNA array version 1.0 (12×135k; Arraystar, Shanghai, China), containing probes for 18,534 lncRNAs and 18,874 coding transcripts (collected from databases such as NCBI RefSeq, UCSC Known Genes, NRED, RNAdb and Ensembl), was used for detection. Total RNA (~5 μg) from each sample was used for labeling and array hybridization with the following steps: i) Reverse transcription with a SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen); ii) double-stranded cDNA labeling with a NimbleGen One-Color DNA Labeling kit (Roche, Mannheim, Germany); iii) array hybridization using the NimbleGen Hybridization System (Roche), followed by washing with the Nimblegen Wash Buffer kit (cat. no. 05584507001; Roche); and iv) array scanning using the Axon GenePix 4000B Microarray scanner (Molecular Devices). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5; Roche) for grid alignment and expression data analysis. Additionally, hierarchical clustering was performed to present distinguishable mRNA expression profiling among samples.
3
Bacillus subtilis Transcriptome Profiling via Custom Microarray
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Per sample, 100 ng of total RNA was combined with Array Control RNA spikes (Ambion) and labeled using a low input Quick Amp whole-transcript (WT) labeling kit (Agilent) according to the manufacturer’s instructions. Each hybridization mixture comprised 1.1 μg test (Cy3) and 1.1 μg reference (Cy5) components, representing a common reference pool of all samples. The samples were dried, and 1.98 μl water was added. The hybridization cocktail was made according to the manufacturer’s instructions (NimbleGen Arrays User’s Guide—Gene Expression Arrays Version 5.0, Roche NimbleGen). Then, 7.2 μl from this mix was added to each sample. The samples were incubated for 5 min at 65°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto a 12-by-135,000 custom microarray designed against Bacillus subtilis (Roche NimbleGen, Inc.) as used in previous studies (4 (link), 46 (link)). Microarrays were hybridized for 20 h at 42°C with a NimbleGen hybridization system (Roche NimbleGen, Inc.). Afterward, the slides were washed according to the NimbleGen Arrays User’s Guide—Gene Expression Arrays Version 6.0 and scanned with an Agilent G2565CA DNA microarray scanner (Agilent Technologies).
4
Genome-wide Gene Expression Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using either TRIzol reagent (Invitrogen) or an RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturers' protocols. Following quality confirmation, RNA samples were amplified and labeled using a NimbleGen One Color DNA Labeling kit (Roche, Madison, WI, USA). RNA was then hybridized (NimbleGen Hybridization System, Roche, Madison, WI, USA) to a human genome array (12 × 135 K array; Roche) containing oligos representing 45,033 human genes. After washing, the processed slides were scanned with an ELx800 absorbance microplate reader (Biotek Instruments Inc., Winooski, VA, USA). NimbleScan (version 2.6., Roche NimbleGen, Madison, WI, USA) was used for data analysis, including quantile normalization and background correction. Agilent GeneSpring software (Agilent Technologies, Santa Clara, CA, USA) was used to further analyze the gene summary files. Three samples were randomly selected from each group for microarray analysis. The genes were considered differentially expressed when the ratio was >1.5. or <0.6.7 in normalized intensity between the two groups or when P-values were less than 0.0.5 by one-way analysis of variance (ANOVA).
5
Construction of Secondary Genotyping Array
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To select probes for the construction of a secondary array, CS, M808, and 14 randomly selected RILs were genotyped. After digestion of total DNA with PstI and BstNI, adapter ligation, PCR amplification and purification of PCR products as described in the library preparation section, they were labelled with Cy3 using the NimbleGen One-Color DNA Labeling Kit (Roche Diagnostics) according to the manufacturer's instructions. Hybridization was performed at 42°C for 72 h on a NimbleGen Hybridization System (Roche Diagnostics). Arrays were scanned using a NimbleGen MS200 Microarray Scanner (Roche Diagnostics) and genotype calling was performed based on the signal intensities.
For selection of probes, a χ2 test was used to compute segregation of each probe in the 14 RILs. Those deviating from Mendelian 1 : 1 segregation at a 1% significance level were discarded. The secondary array was constructed using one selected probe per contig or read, consisting of 21,346 CS-derived probes, 21,205 M808-derived probes, and 2,303 probes for normalization. These 44,854 probes were spotted in triplicate on a NimbleGen 12 × 135 K array (Roche Diagnostics). This array was used for genotyping of 210 RILs and the two parental lines.
For selection of probes, a χ2 test was used to compute segregation of each probe in the 14 RILs. Those deviating from Mendelian 1 : 1 segregation at a 1% significance level were discarded. The secondary array was constructed using one selected probe per contig or read, consisting of 21,346 CS-derived probes, 21,205 M808-derived probes, and 2,303 probes for normalization. These 44,854 probes were spotted in triplicate on a NimbleGen 12 × 135 K array (Roche Diagnostics). This array was used for genotyping of 210 RILs and the two parental lines.
6
Genotyping of F1 Progeny Using HD-2 135K Microarray
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genotypes of the parental plants, P1 and P2, and their 178 F1 progeny were determined by using HD-2 135K × 12plex microarrays as described. After digestion of total DNA with PstI and BstNI, adapter ligation, PCR amplification and purification of PCR products, as described in library preparation section, they were labelled with Cy3 using NimbleGen One-Color DNA Labeling Kit (Roche Diagnostics) according to the manufacturer’s instructions. Hybridization was performed at 42°C during 24 hours on NimbleGen Hybridization System (Roche Diagnostics). The microarrays were scanned with a NimbleGen MS200 Microarray Scanner (Roche Diagnostics) and genotype calling was performed based on the signal intensity. Present/absent calls of the probe-sets reflected the present/absent DNA fragments hybridized to the probe-sets. Thus, DNA polymorphisms genotyped with this system were used as dominant markers.
7
Microarray Analysis of Testis Transcriptome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microarray experiments were conducted by KangChen Bio-technology Co. (Shanghai, China) according to the standard procedure [11 (link)]. Briefly, total RNAs from testes were extracted using Trizol (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturers’ instructions. Genomic DNA contamination was removed by DNase treatment. The concentration of RNA was measured through a Nanodrop ND-1000 Spectrophotometer (NanoDrop products, Wilmington, DE, USA). Precisely 10 mg of total RNA was taken to synthesize double-stranded cDNA, following the steps of an Invitrogen Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen). RNA contamination was removed by RNase A, and cDNA was precipitated and labelled using a NimbleGen One-Color DNA Labeling Kit (RocheNimbleGen, Inc., Pleasanton, CA, USA). Labelled cDNA was quantitated and hybridized with microarray chip slides in the NimbleGen Hybridization System (Roche-NimbleGen, Inc.). Following hybridization and washing, the microarray slides were scanned with an Axon GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA).
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!