Total RNA was extracted from plasma EVs and liver tissues using the 3D-Gene RNA extraction reagent (Toray, Kamakura, Japan) and the miRNeasy Mini Kit (Qiagen, Hilden, Germany), respectively, according to the manufacturers’ instructions. The quality of the extracted total RNA was checked using a Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). A comprehensive miRNA microarray analysis was performed using 3D-Gene Human miRNA Oligo chips V22 (Toray) designed to detect 2632 miRNAs registered in the miRBase (release 22) database31 (link)–33 (link). To normalize the signals, three preselected internal control miRNAs (miR-149-3p, miR-2861, and miR-4463) were used for EVs34 (link), whereas the global median normalization method was used and the median distribution was set at 25 for liver tissues.
3d gene rna extraction reagent
Manufactured by Toray
Sourced in Japan
The 3D-Gene RNA extraction reagent is a laboratory product designed for the extraction and purification of RNA from various sample types. It functions to isolate and concentrate RNA molecules, preparing them for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
3d gene human mirna oligo chip, by Toray (18 mentions)
3d gene mirna labeling kit, by Toray (15 mentions)
3d gene extraction software, by Toray (5 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
3d gene scanner 3000, by Toray (3 mentions)
Liquid sample kit, by Toray (2 mentions)
3d gene human mirna oligo chip ver 17.0, by Toray (2 mentions)
3d gene scanner, by Toray (2 mentions)
Rneasy 96 qiacube ht kit, by Qiagen (2 mentions)
3d gene human mirna oligo chip version 21, by Toray (2 mentions)
3d gene mirna labelling kit, by Toray (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Agilent rna 6000 pico kit, by Agilent Technologies (2 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
3d gene microrna labeling kit, by Toray (1 mentions)
3d gene human microrna oligo chip, by Toray (1 mentions)
Advanced dmem, by Thermo Fisher Scientific (1 mentions)
Multi bead shocker, by Yasui Kikai (1 mentions)
Genespring gx, by Agilent Technologies (1 mentions)
Mircury lna microrna array power labeling kit, by Qiagen (1 mentions)
30 protocols using 3d gene rna extraction reagent
1
Comprehensive miRNA Profiling in Plasma EVs
2
Microarray Analysis of Serum miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total miRNA was extracted from 300 μl aliquots of serum samples using the 3D-Gene® RNA extraction reagent from a liquid sample kit (Toray Industries, Inc., Kanagawa, Japan). Comprehensive miRNA expression analysis was performed using a 3D-Gene® microRNA Labeling kit and a 3D-Gene® Human microRNA Oligo Chip (Toray Industries, Inc.), designed to detect 2540 miRNA sequences registered in miRBase releases 20 and 21 (http://www.mirbase.org/ ). MiRNA was defined as present if the corresponding microarray signal was more than the signal (mean + 2 × standard deviations) of the negative controls, of which the top and bottom 5%, ranked by signal intensity, were considered noise generated by the experimental system and were removed. When the signal value was negative, or undetected, after background subtraction, the value was replaced by 0.1 on a base 2 logarithm scale. Three pre-selected internal control miRNAs (miR-149-3p, miR-2861, and miR-4463) were used to normalize signals across the different microarrays tested. The results were subjected to probability analysis using receiver operating characteristic curves. All microarray data of this study are in agreement with the Minimum Information About a Microarray Experiment (MIAME) guidelines and are publicly available through the GEO database (GSE134108, http://www.ncbi.nlm.nih.gov/projects/geo/ ).
3
Serum and Vitreous miRNA Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from serum and vitreous samples using miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. Gene tip miRNA was extracted from the serum and vitreous samples using 3D-Gene®RNA extraction reagent from a liquid sample kit (Toray Industries, Inc., Kamakura, Kanagawa, Japan) and concentrated. Fluorescent labeling of RNA was performed using 3D-Gene® miRNA Labeling kit. Labeled RNA was hybridized to a 3D-Gene® Human miRNA Oligo Chip (Toray Industries, Inc.) designed to detect 2565 mature human miRNA sequences registered in miRBase release 21 (http://www.mirbase.org/ ). The chip was scanned using a 3D-Gene® Scanner, miRNAs with signals higher than the background signal were selected (positive call), and only miRNAs with positive call were used in subsequent analyses. The miRNA signal values were standardized by global normalization (log conversion of data and median alignment) [22 (link)].
4
Serum miRNA Profiling via Fluorescent Microarray
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene tip miRNA was extracted from fresh frozen serum samples using the 3D-Gene RNA extraction reagent from a liquid sample kit (Toray Industries, Inc., Kanagawa, Japan) and concentrated. The extracted miRNA was florescent labeled using the 3D-Gene miRNA Labeling kit (Toray Industries, Inc.). The florescent labeled RNA was hybridized to a 3D-Gene Human miRNA Oligo Chip (Toray Industries, Inc.)19 (link) designed to detect 2565 mature human miRNA sequences registered in miRBase Release 21 (http://www.mirbase.org/ ). The chip was scanned using a 3D-Gene Scanner. The miRNAs with signals higher than the background signal were first selected (positive call), and the background signal was subtracted from each positive call miRNA signal. MicroRNA signal values were standardized by global normalization as follows. For each sample, raw data were log-transformed, and the median was calculated. All data were shifted so that the median values were aligned, except when raw data = 2; in that case, the data were not shifted.
5
Exosome Isolation and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown in 10-cm plates for 48 hours beforehand, then cells and culture supernatant were collected. The medium was replaced with either “advanced DMEM” (Thermo Fisher Scientific) or RPMI containing an antibiotic-antimycotic mixture and 2 mM L-glutamine (not containing fetal bovine serum), and incubated for 48 hours. Approximately 6 × 104 cells and 1.5 mL cell culture supernatant (into which extracellular particles such as exosomes were released) were collected.
Exosomes were prepared by further extraction from the cell culture supernatant; cells and cell debris were removed by centrifugation at 2,000 × g for 10 minutes at 4°C and filtration, followed by further centrifugation at 110,000 × g for 70 minutes at 4°C. The pellets were washed and resuspended in 11 mL phosphate-buffered saline, and centrifuged again at 110,000 × g for 70 minutes at 4°C [13 (link)]. Finally, the pellet (exosomes) was resuspended in 300 μL phosphate-buffered saline.
Total RNA derived from the cell culture supernatant or exosomes was extracted using the 3D-Gene RNA extraction reagent (Toray Industries, Inc., Japan), whereas total RNA derived from cells was extracted using the miRNeasy Mini kit (QIAGEN, Hilden, Germany, catalog #217004). (dx.doi.org/10.17504/protocols.io.vu3e6yn )
Exosomes were prepared by further extraction from the cell culture supernatant; cells and cell debris were removed by centrifugation at 2,000 × g for 10 minutes at 4°C and filtration, followed by further centrifugation at 110,000 × g for 70 minutes at 4°C. The pellets were washed and resuspended in 11 mL phosphate-buffered saline, and centrifuged again at 110,000 × g for 70 minutes at 4°C [13 (link)]. Finally, the pellet (exosomes) was resuspended in 300 μL phosphate-buffered saline.
Total RNA derived from the cell culture supernatant or exosomes was extracted using the 3D-Gene RNA extraction reagent (Toray Industries, Inc., Japan), whereas total RNA derived from cells was extracted using the miRNeasy Mini kit (QIAGEN, Hilden, Germany, catalog #217004). (
6
GJA1 SNP and Serum miRNA Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We investigate the relationships between the GJA1 SNP rs1015451 and serum concentrations of 2555 miRNAs. The total RNA was extracted from individual serum samples using the 3D-Gene RNA Extraction Reagent from a liquid sample kit (Toray Industries, Inc., Kanagawa, Japan). A total of 2555 miRNA sequences were detected using the 3D-Gene miRNA Labeling kit and 3D-Gene Human miRNA Oligo Chip (Toray Industries, Inc). We analyzed the relationships between the GJA1 SNP rs1015451 genotype and serum concentrations of the miRNAs in the AF patients24 (link).
7
Comprehensive miRNA Profiling of Serum and Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum RNA was extracted from 300 µl of serum using the 3D-Gene® RNA extraction reagent (Toray Industries, Inc.). Fresh-frozen tissues were crushed to a powder using a Multibead Shocker (Yasui Kikai Corporation) under liquid nitrogen (−196°C). Total RNA was extracted from frozen tumor tissue powder using the miRNeasy Mini kit (cat. no. 217004; Qiagen GmbH). Comprehensive miRNA expression analysis was performed using the 3D-Gene® miRNA Labeling kit and the 3D-Gene® Human miRNA Oligo Chip (both Toray Industries, Inc.), which was designed to detect 2,588 miRNA sequences registered in miRBase release 21 database (http://www.mirbase.org/ ). Microarray experiments were performed by Kamakura Techno-Science Inc. miRNAs with a signal intensity >26 were considered detected miRNAs. Principal component analysis (PCA) map and heatmap were generated by Genomics Suite version 6.6 (Partek Inc.).
8
Plasma miRNA Profiling by Microarray
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 300 μL of the frozen plasma using a 3D-Gene® RNA extraction reagent (TORAY Industries, Inc., Tokyo, Japan), and half of the extracted RNA was used for analysis. MiRNAs were labeled using Exiqon miRCURY LNA™ microRNA Array Power Labeling kit (Exiqon Inc., Woburn, MA, USA) and were analyzed with one-channel microarray using the 3D-Gene® Human miRNA oligo chip (Ver. 17.0) (TORAY Industries, Inc., Tokyo, Japan), which can analyze the expression of 1720 miRNAs.
From the output fluorescent signals, we subtracted the background noise and performed calculations using the global normalization method with a median value of 25.
From the output fluorescent signals, we subtracted the background noise and performed calculations using the global normalization method with a median value of 25.
9
Comprehensive miRNA Analysis of Amniotic Fluid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 300 μl of amniotic fluid using the 3D-Gene® RNA extraction reagent (Toray Industries, Inc., Tokyo, Japan). Half of the extracted RNA was used for comprehensive miRNA expression analysis using a 3D-Gene miRNA labeling kit and a 3D-Gene Human miRNA Oligo Chip (Toray). Scanning was performed on a 3D-Gene Scanner 3000 (Toray). The 3D-Gene extraction software (v. 1.2, Toray) was used to read the raw intensity of the image. The raw data were analyzed using GeneSpringGX (v. 10.0, Agilent Technologies, CA, USA) and quartile normalized. The microarray data from this study have been deposited at the NCBI Gene Expression Omnibus with the accession number GSE143193.
10
Plasma miRNA Profiling in sALS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was collected in disodium ethylenediaminotetraacetate (Na2EDTA) tubes and centrifuged immediately, after which the plasma was separated and frozen at −80 °C. RNA (total RNA) was extracted from 300 μl of the frozen plasma using a 3D-Gene® RNA extraction reagent (TORAY Industries, Inc., Tokyo, Japan) and the half of the extracted product was used. After being labeled with an Exiqon miRCURY LNA™ microRNA Array Power Labeling kit (Exiqon Inc., Woburn, MA, USA), miRNAs were analyzed with a 3D-Gene® Human miRNA oligo chip (Ver. 17.0) (TORAY Industries, Inc., Tokyo, Japan) which carries approximately 1800 probes to detect human miRNAs using fluorescent signals. From the outputted signal data, we subtracted the background noise, and calibrated using the global normalization method with the median value as 25.
Comparisons of all normalized miRNA data calculated as a binary logarithm underwent a paired t test, Wilcoxon rank sum test, fold change ratio and variance using JMP® Pro 11.0.0. (SAS Institute Inc., Cary, North Carolina, USA). MiRNA showing the most significant changes either up-regulating or down-regulating in sALS patients, were chosen as the biomarker candidates for validation analysis.
Comparisons of all normalized miRNA data calculated as a binary logarithm underwent a paired t test, Wilcoxon rank sum test, fold change ratio and variance using JMP® Pro 11.0.0. (SAS Institute Inc., Cary, North Carolina, USA). MiRNA showing the most significant changes either up-regulating or down-regulating in sALS patients, were chosen as the biomarker candidates for validation analysis.
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!