Total RNA was extracted from three tumor tissues and paired normal colorectal tissues using miRNeasy Mini kit (Qiagen, Valencia, CA, U.S.A.). Purity and quantity of total RNA were evaluated by NanoDrop ND-1000 Spectrophotometry (Thermo Scientific, U.S.A.) and Agilent’s 2100 Bioanalyzer. miRNA microarray profiling was performed as previously described [13 (link)]. Data analysis was performed using GeneSpring GX software (Agilent). Finally, the heat map of the 57 microRNAs most obvious differences was created using a method of hierarchical clustering by GeneSpring GX, version 7.3 (Agilent Technologies, California, United States).
Nanodrop nd 1000 spectrophotometry
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The NanoDrop ND-1000 is a spectrophotometry instrument designed for the measurement of small sample volumes. It utilizes a patented sample retention system to enable the analysis of 1-2 μL samples without the need for cuvettes or other sample containment devices. The NanoDrop ND-1000 provides rapid and accurate quantification of nucleic acid and protein concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Genespring gx version 7, by Agilent Technologies (4 mentions)
Mirneasy mini kit, by Qiagen (3 mentions)
Rnaiso plus reagent, by Takara Bio (3 mentions)
Dnase 1, by New England Biolabs (3 mentions)
Agilent s 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Superscript 2 rt, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Axon genepix 4000b microarray scanner, by Molecular Devices (2 mentions)
Genepix pro 6.0 program, by Molecular Devices (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Genespring software version 7, by Agilent Technologies (2 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (2 mentions)
Taqman microrna rt kit, by Thermo Fisher Scientific (2 mentions)
Rt preamp primers, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 7900ht real time pcr instrument, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Mirneasy micro kit, by Qiagen (2 mentions)
Genespring gx software, by Agilent Technologies (1 mentions)
Trizol rna extraction kit, by Thermo Fisher Scientific (1 mentions)
Mrna only eukaryotic mrna isolation kit, by Illumina (1 mentions)
26 protocols using nanodrop nd 1000 spectrophotometry
1
Colorectal Cancer miRNA Profiling
2
Vitreous Humor RNA Extraction
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RNA was isolated from vitreous humor samples using a Trizol RNA extraction kit (Invitrogen, Carlsbad, CA, USA). The integrity and quantity of each RNA sample were assessed by agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA).
3
One-Color Microarray-Based Gene Expression Analysis
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Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs were measured by NanoDrop ND-1000 spectrophotometry (Thermo Scientific). 1μg of each labeled cRNA was fragmented by adding 5μl 10× blocking agent and 1μl of 25× fragmentation buffer, then heated the mixture at 60°C for 30 min, finally 25μl 2× GE Hybridization buffer was added to dilute the labeled cRNA. 50μl of hybridization solution was dispensed into the gasket slide and assembled to the lncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned using the Agilent Microarray Scanner.
4
RNA Extraction and RT-qPCR Analysis
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Total RNA of cells or corneas were extracted by the RNA iso plus reagent (TaKaRa, Dalian, China), and the RNA was quantified by the NanoDrop ND-1000 Spectrophotometry (Thermo Fisher Scientific). The first strand cDNA was synthesized by a two-step method using PrimeScript RT reagent Kit from total RNA. Then cDNA was used for PCR in 20-µL reaction volume following the manufacturer's instructions. The PCR program for the reactions was described as before.31 (link) The ΔΔCT method was used for analysis of target gene products. Data are expressed as fold of increase in mRNA expression. The primer pair sequences are listed in the Table .
5
RNA Extraction and cDNA Synthesis from Plant Leaves
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Leaf material (2.5 cm2 area) from pathogen-inoculated and non-inoculated controls was collected at 3 and 12 days, flash frozen in liquid nitrogen and stored at −80° C. Total RNA was extracted from leaf tissues using the Concert® Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), with purification using the INVISORB Spin Plant RNA Mini Kit (Invitek, Hayward, CA, USA), according to manufacturer’s instructions. A total of 1 μg of each total RNA was treated with 2 U of DNase I (New England Biolabs, Ipswich, MA., USA) for elimination of residual DNA. Analysis of total RNA concentration and integrity was conducted following 1% agarose gel electrophoresis and Nanodrop ND-1000 spectrophotometry (Thermo Fischer Scientific, Waltham, MA, USA).
For cDNA synthesis, pools containing equimolar amounts of total RNA from three biological replicates were prepared. In total, eight pools were prepared, one for each experimental condition (Table1 ). A total of 1 μg of total RNA was reverse transcribed to cDNA using Super Script II RT and Oligo(dT)16–18 primers (Invitrogen, Carlsbad, CA, USA).
For cDNA synthesis, pools containing equimolar amounts of total RNA from three biological replicates were prepared. In total, eight pools were prepared, one for each experimental condition (Table
6
Profiling miRNA Expression in Spinal Cord
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Total RNA was isolated from spinal cord tissues using RNAEasy plus kit (Qiagen) according to the manufacturer’s protocol. The RNA was quantified by NanoDrop ND-1000 spectrophotometry (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). Total RNA (200 ng) was labeled with fluorescence dye hy3 or hy5 using the miRCURY Hy3/Hy5 Power Labeling kit and hybridized on the miRCURY™ LNA array (v.16.0; Exiqon A/S, Copenhagen, Denmark), which were designed based on miRBaserelease 10.0 and contained 546 probes from humans, mice and rats. The procedure and imaging processes were as described previously [29 (link)].
7
MicroRNA Array Analysis in Diabetic Nephropathy
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The microRNA array was performed by Kangcheng Bio-Tech, Inc. Total RNA was isolated from peripheral blood of patients with DN and controls using a miRNeasy kit (Qiagen, Inc.). The RNA purity was determined via NanoDrop ND-1000 spectrophotometry (Thermo Fisher Scientific, Inc.) and the RNA quality was determined using 1% agarose-formaldehyde denaturing gel electrophoresis. After RNA quantitation, the samples were assessed using the miRCURY LNA™ Array v. 16.0 (Exiqon A/S; Qiagen, Inc.) according to the manufacturer's protocol. The procedure and imaging processes were performed as described previously (16 (link)). The microarray data were analyzed using Agilent Feature Extraction software (version 10.7; Agilent Technologies, Inc.) (17 (link)). Differentially expressed miRNAs were screened with an unpaired t-test (P<0.05) combined with a significant threshold value of a fold change [FC; (log2 (FC) >2 for upregulated, and log2 (FC) ≤-2 for downregulated]. The microarray data that support the findings of this study are available from the corresponding author upon reasonable request.
8
Total RNA Extraction from Plant Leaves
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Total RNA was extracted from inoculated and non-inoculated leaf fragments of C4 and CAV plants using the PureLink® Plant RNA Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and subsequently purified using the Invitrap® Spin Plant RNA Mini Kit (Invitek, Hayward, CA, USA), following the manufacturer’s instructions. To avoid the presence of DNA, samples were treated with 2U of DNase I (New England Biolabs, Ipswich, MA, USA). RNA concentration and integrity were evaluated by agarose gel electrophoresis and Nanodrop ND-1000 spectrophotometry (Thermo Scientific, Waltham, MA, USA), based on 260/280 nm ratio values.
9
Profiling Oral Cancer miRNA Expression
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Total RNA was extracted from the OSCC tissues using an miRNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA). The purity and quantity of the total RNA were evaluated via NanoDrop ND-1000 spectrophotometry (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the Agilent 2100 Bioanalyzer. Total RNA (200 ng) was labeled with fluorescence dye hy3 or hy5 using the miRCURY Hy3/Hy5 Power Labeling kit and hybridized on the miRCURY™ LNA Array (v.16.0), both obtained from Exiqon; Qiagen, Inc., according to the manufacturer’s protocol. Following washing with PBS, the Axon GenePix 4000B microarray scanner (Axon Instruments; Molecular Devices, LLC, Sunnyvale, CA, USA) was used to scan the fluorescence intensity of the microarray. The scanned images were then imported into the GenePix Pro 6.0 program (Axon Instruments; Molecular Devices, LLC) for grid alignment and data extraction. Finally, the heat map of the 57 miRNAs with the most marked differences was created using a method of hierarchical clustering in GeneSpring GX software, version 7.3 (Agilent Technologies, Inc., Santa Clara, CA, USA).
10
Corneal RNA Extraction and qRT-PCR Analysis
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Total RNA of corneas were extracted by the RNAiso plus reagent (TaKaRa, Dalian, China), and the RNA was quantified by a NanoDrop ND-1000 Spectrophotometry (Thermo Fisher Scientific) according to the manufacturer's instructions as described earlier.30 (link) The extracted mRNA (n = 5/group/time) was used as the template, and cDNA productions (1 µg) were synthesized by a two-step method using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). PCR was assayed in 20 µL reaction system (2 µL of diluted cDNA (1:12.5), 10 µL of SYBR Premix Ex TaqTM (TaKaRa), 1 µL of diluted primers (1:9), and 7 µL DEPC-treated water). The PCR program for the reactions was described as before.31 (link) Analysis of target genes was quantified by the 2ΔΔCT method.32 (link) The corresponding primers are listed in the Table .
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