Agilent low rna input linear amplification kit

Manufactured by Agilent Technologies

Sourced in United States

The Agilent Low RNA Input Linear Amplification kit is a laboratory equipment product designed for linear amplification of small amounts of RNA. The kit provides a method to generate amplified RNA from limited starting material without introducing bias.

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Lab products found in correlation

Rneasy spin column, by Qiagen (1 mentions) Isogen, by Nippon Gene (1 mentions) Feature extraction software version 9, by Agilent Technologies (1 mentions) Rneasy mini kit, by Agilent Technologies (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Cy3 ctp, by PerkinElmer (1 mentions) Cy5 ctp, by PerkinElmer (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Feature extraction software, by Agilent Technologies (1 mentions) Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Rneasy minelute cleanup kit, by Qiagen (1 mentions) Genepix 4000b microarray scanner, by Molecular Devices (1 mentions) Genepix pro 6, by Molecular Devices (1 mentions)

4 protocols using agilent low rna input linear amplification kit

Ovarian Tissue RNA Extraction and Microarray

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The total RNA for microarray analysis was extracted from the ovarian tissue using ISOGEN (Nippon Gene, Tokyo, Japan) in accordance with the manufacturer’s protocol. ISOGEN is a phenol-based pre-made reagent for RNA extraction.
Complementary DNA was prepared from 500 ng of total RNA from each replicate, as described in the manual for Agilent Low RNA Input Linear Amplification kit (Agilent Technologies, Palo Alto, CA, USA). Double-stranded cDNA was synthesized using the kit, and Cy3-labeled cRNA was prepared by cDNA in vitro transcription in the presence of cyanine 3-CTP dyes. Fluorescently labeled RNA was then purified with Qiagen RNeasy spin columns in accordance with the manufacturer’s protocol (Qiagen, Hilden, Germany). After purification, the cRNA was stored at –80 °C until use.

Klangnurak W, & Tokumoto T. (2017). Fine selection of up-regulated genes during ovulation by in vivo induction of oocyte maturation and ovulation in zebrafish. Zoological Letters, 3, 2.

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Microarray Analysis of Yeast mRNA

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Messenger RNA (mRNA) was isolated and hybridized to Agilent yeast microarrays as described in (46 (link)). Briefly, 5-ml cultures were collected on filters and snap frozen in liquid nitrogen. Total RNA was extracted using the Qiagen RNeasy Mini kit, including the additional DNase I digestion step. Chromosomal RNA (cRNA) for microarray hybridization was synthesized following the standard protocol of the Agilent Low RNA Input Linear Amplification kit (Agilent Technologies). cRNA was extracted using the Qiagen RNeasy Mini kit and hybridized to Agilent Yeast Gene Expression Microarray (8 × 15K G4813A) slides and scanned at 5-μm resolution. Data were extracted using Agilent Feature Extraction software version 9.5 with Linear Lowess dye normalization and no background subtraction and were submitted to the Princeton University Microarray database for storage and analysis.
For estradiol induction experiments, time course fold change in transcript levels was fit to a Hill plot by optimization of n, f₀, K and V_max for each gene for the equation f(t) = f₀ + V_max·tⁿ/(Kⁿ + tⁿ). Delay times were determined by extrapolation of the derivative of this function at f(t) = V_max/2 to the x-axis intercept.

Elfving N., Chereji R.V., Bharatula V., Björklund S., Morozov A.V, & Broach J.R. (2014). A dynamic interplay of nucleosome and Msn2 binding regulates kinetics of gene activation and repression following stress. Nucleic Acids Research, 42(9), 5468-5482.

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RNA Extraction and Expression Profiling

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RNA from each of the 69 cell lines was extracted using TRIZOL Reagent following the manufacturer's recommendations (Invitrogen, Burlington, ON). RNA was purified using the RNeasy MinElute Cleanup Kit (Qiagen, Mississauga, ON). RNA quality was assessed by Agilent 2100 Bioanalyzer (Version B.02.02). RNA with an integrity number of at least 7 was amplified and labeled using the Agilent Low RNA Input Linear Amplification kit (Agilent, Santa Clara, CA). Labeling reactions were performed with 250 ng total RNA, along with the Agilent Spike-in RNA mix, using Cy3-CTP and Cy5-CTP for control (-IR) and experimental (+IR) RNA, respectively (Perkin Elmer, MA, USA). Amplified RNA was quantified using the NanoDrop ND-1000 (NanoDrop Technologies, DE, USA) and the concentration of cRNA and the specific dye activity were calculated. Samples with a specific dye activity greater than 8 pmol/µl were selected for hybridization to arrays. Pairs of cRNA (unirradiated versus irradiated) were hybridized to Agilent Whole Human Genome Oligo 4x44K GE arrays as per the product protocol. Image acquisition and analysis were done using an Agilent Microarray Scanner, Model G2565BA and Agilent Feature Extraction software v9.1 set to default settings. Raw data has been submitted to the NCBI GEO database (Accession Number GSE19541.)

Feilotter H.E., Michel C., Uy P., Bathurst L, & Davey S. (2014). BRCA1 Haploinsufficiency Leads to Altered Expression of Genes Involved in Cellular Proliferation and Development. PLoS ONE, 9(6), e100068.

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Maize cDNA Microarray Expression Analysis

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cDNA was synthesized from total RNA using a Agilent Low RNA Input Linear Amplification Kit, with spike-in controls (5184-3523), following the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA). Two of the four control samples and two of the four treatment samples were labelled with Cy3 dye and the other samples were labelled with Cy5 dye, to account for any possible dye-bias that may occur during hybridization or fluorescence detection during scanning of the slides. Agilent 4×44K oligonucleotide two-colour cDNA Maize microarray slides were used. Hybridization and washing was performed following the Agilent Two-Colour Microarray-Based Gene Expression Analysis protocol (Agilent Technologies). A total of four 4×44k slides (i.e. four technical replicates) were used for each of the four biological replicates. Hybridized slides were scanned with a GenePix4000B microarray scanner (Axon Instruments, Concord, ON, Canada) and features were extracted and analysed using GenePix Pro 6.1 (Axon Instruments). Each array was inspected individually and low-quality spots (i.e. those with fluorescence levels that were not significantly distinguishable from the background fluorescence in the red-to-green channel intensity ratio histograms of the array) were manually flagged and eliminated from further analysis.

Spence A.K., Boddu J., Wang D., James B., Swaminathan K., Moose S.P, & Long S.P. (2014). Transcriptional responses indicate maintenance of photosynthetic proteins as key to the exceptional chilling tolerance of C4 photosynthesis in Miscanthus × giganteus. Journal of Experimental Botany, 65(13), 3737-3747.

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