Low rna input linear amp kit

Manufactured by Agilent Technologies

Sourced in United States

The Low RNA Input Linear Amp Kit is a laboratory equipment product designed for linear amplification of low input RNA samples. The kit enables users to generate sufficient quantities of amplified RNA from limited starting material for downstream analysis.

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6 protocols using low rna input linear amp kit

Microarray Analysis of Mouse RNA

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Total RNA was purified by using RNeasy Mini kit (QIAGEN). Microarray targets from 200 ng total RNA were synthesized and labeled using the Low RNA Input Linear Amp Kit (Agilent) and hybridized to Mouse 4×44K Ver.2.0 arrays. Arrays were scanned on an Agilent Technologies Microarray scanner, and signal intensities were calculated using the Agilent Feature Extraction 10.7.3.1 software. GEO accession number is GSE59107.

Ohinata Y, & Tsukiyama T. (2014). Establishment of Trophoblast Stem Cells under Defined Culture Conditions in Mice. PLoS ONE, 9(9), e107308.

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Liver Transcriptome Profiling Protocol

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For the expression analysis of human liver cultures, total RNA was isolated from liver biopsies or from organoid cultures grown in our defined medium, using QIAGEN RNAase kit following the manufacturer’s instructions. Five hundred ng of total RNA were labeled with low RNA Input Linear Amp kit (Agilent Technologies). Universal human reference RNA (Agilent) was differentially labeled and hybridized to the tissue or cultured samples. A 4X 44 K Agilent whole human genome dual color microarray (G4122F) was used. Labeling, hybridization, and washing were performed according to Agilent guidelines. Microarray signal and background information were retrieved using Feature Extraction software (V.9.5.3, Agilent Technologies). Hierarchical clustering analysis was performed in whole-liver tissue or organoid arrays. A cut-off of 3-fold differentially expressed was used for the clustering analysis.

Huch M., Gehart H., van Boxtel R., Hamer K., Blokzijl F., Verstegen M.M., Ellis E., van Wenum M., Fuchs S.A., de Ligt J., van de Wetering M., Sasaki N., Boers S.J., Kemperman H., de Jonge J., Ijzermans J.N., Nieuwenhuis E.E., Hoekstra R., Strom S., Vries R.R., van der Laan L.J., Cuppen E, & Clevers H. (2015). Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver. Cell, 160(1-2), 299-312.

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RNA Extraction and Gene Expression Analysis

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Cells were harvested with lysis buffer as described above to extract total RNA. Quality of RNA was controlled by the Agilent 2100 Bioanalyzer platform (Agilent Technologies, Santa Clara, CA, USA); RNA Integrity Number (RIN) was between 9.8 and 10. Amplification and labelling (Cy3 and Cy5) were performed using the Low RNA Input Linear Amp Kit (Agilent Technologies, Santa Clara, CA, USA). Hybridization was conducted on Agilent Whole Genome Oligo (60-mer) 4 × 44K microarrays with the Agilent Gene Expression Hybridization Kit. Agilent’s feature extraction Software was used to determine spot intensities and Cy5/Cy3 ratios after background subtraction with ratios displaying the expression level in KD compared to control in a logarithmic scale. Mean fold changes were calculated from four replicate measurements. The heat map was created by Gene Spring Software (Agilent Technologies, Santa Clara, CA, USA).

Zilleßen P., Celner J., Kretschmann A., Pfeifer A., Racké K, & Mayer P. (2016). Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes. Scientific Reports, 6, 23074.

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Transcriptomic Analysis of ATRA Treatment

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Total RNA was isolated from the cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) at 0, 0.5, and 1 h after the ATRA treatment. The quality of the extracted RNA was checked using a Bioanalyzer 2100, RNA 6000 Nano LabChips, and the 2100 Expert standard software (all Agilent Technologies, Santa Clara, CA, USA). To prepare cDNA, approximately 0.5 g of each RNA sample was used in the reaction of the reverse transcription, performed using the Low RNA Input Linear Amp Kit (Agilent Technologies, Santa Clara, CA, USA) according to standard protocol. The cRNA samples for time points 0, 0.5, and 1 h after the ATRA treatment were labeled with Cy5-CTP (Perkin Elmer, Waltham, MA, USA) and the control samples (the time point 0 h) were labeled with Cy3-CTP (Perkin Elmer, Waltham, MA, USA). The cRNA fragmentations and hybridizations were performed using in situ the Hybridization Kit Plus (Agilent Technologies, Santa Clara, CA, USA) according to standard protocol. Data acquisition was performed using the DNA Microarray Scanner G2505C (Agilent Technologies, Santa Clara, CA, USA). The raw transcriptome data were processed using the Feature Extraction software (version 10.1.3.1; Agilent Technologies, Santa Clara, CA, USA).
Statistical data analysis by ANOVA with the p-value cut-off set at 0.05 was carried out using the GeneSpring GX12.5 software (Agilent Technologies, Santa Clara, CA, USA).

Novikova S., Tolstova T., Kurbatov L., Farafonova T., Tikhonova O., Soloveva N., Rusanov A., Archakov A, & Zgoda V. (2022). Nuclear Proteomics of Induced Leukemia Cell Differentiation. Cells, 11(20), 3221.

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DNA Microarray Hybridization Protocol

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Conduction of DNA chip hybridization was performed as described previously (Mayer et al. 2012) . In brief, samples were amplified and labeled using Low RNA Input Linear Amp Kit (Agilent Technologies, Santa Clara, CA, USA). Control samples were labeled with Cy3 and samples from stimulated cells with Cy5. DNA chip hybridization was conducted on Agilent Whole Human Genome Oligo (60mer) 4!44 K microarrays corresponding to the 60-mer oligo microarray processing protocol of the Gene Expression Hybridization Kit (Agilent Technologies). For statistical analysis, ratios of expression levels in IR KD vs sh-control cells were calculated and subjected to logarithmic transformation (base 2). Induction in gene expression is signified by a positive and suppression by a negative sign.

Frisch C.M., Zimmermann K., Zilleßen P., Pfeifer A., Racké K, & Mayer P. (2015). Non-small cell lung cancer cell survival crucially depends on functional insulin receptors. Endocrine-related cancer, 22(4).

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Identifying RNA Inosine Modifications

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In order to identify inosines on RNA strands, inosine chemical erasing (ICE) was used 38 with minor modifications. Briefly, total RNA from 100 fly heads was extracted using an RNeasy mini kit (Qiagen. Germany). Then, 10 μg of RNA was cyanoethylated (CE) by incubation in 38 μl solution (50% (v/v) ethanol, 1.1 M triethyl ammonium acetate, pH 8.6) with (CE+) or without (CE-) 1.6 M acrylonitrile at 70°C for 30 min.
After cyanoethylation, RNA was purified using an RNeasy MinElute kit (Qiagen, Germany) and reverse transcribed using Low RNA Input Linear Amp Kit (Agilent Technologies, USA). Sites of nine D. melanogaster genes (Syx1A, Atx2, Atpalpha, CG4587, cpx, EndoA, Alpha-Spec, Cadps, RhoGAP100F) were analyzed by a Sanger sequencing described in the previous section. According to the known method 38 , the editing site in CE-state can be detected as combined signal from A and G in the sequence chromatogram. For CE+ state, the signal can be detected as A. For those sites that are almost 100% edited, no amplification of the cDNA can be detected in the CE+ state 38 .

Kuznetsova K.G., Kliuchnikova A.A., Ilina I.U., Chernobrovkin A.L., Novikova S.E., Farafonova T.E., Karpov D.S., Ivanov M.V., Goncharov A.O., Ilgisonis E.V., Voronko O.E., Nasaev S.S., Zgoda V.G., Zubarev R.A., Gorshkov M.V, & Moshkovskii S.A. (2018). Proteogenomics of Adenosine-to-Inosine RNA Editing in the Fruit Fly. Journal of proteome research, 17(11).

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