Arcturus™ RiboAmp™ PLUS Kit

A single round of amplification yields product sizes ranging from 200 bases to 6 kb. The majority of these products are approximately 1.5 kb in length. A second round of amplification will result in shorter products. We recommend using an Agilent 2100 bioanalyzer to visualize these products. Amplification products can be visualized by agarose gel electrophoresis; they will migrate as a smear. Although this data is still useful, it is less informative than bioanalyzer analysis.

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Glass microarray analysis experiments typically require 5-20 µg of total RNA per slide for sample labeling and hybridization. Thus, microarray-based gene expression analysis of very small samples [laser capture microdissection (LCM), tissue biopsies, or other clinical samples] is difficult due to the very low amounts of total RNA recovered from the samples. Linear amplification of RNA from small samples produces sufficient quantities of RNA for sample labeling and hybridization. Since the amplification technique is highly reproducible and maintains representation of the gene expression in the original sample, it is recommended for probe synthesis by most manufacturers of commercially available microarrays.

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RNA amplification using the Van Gelder and Eberwine technique (Van Gelder 1990) utilizes an oligo(dT) primer containing the T7 RNA polymerase promoter for synthesis of first strand cDNA. The poly(A) tail at the end of mRNA sequences serves as the substrate for the binding of these primers. Since mRNA typically constitutes only 1-5% of the total RNA in the cell, only this fraction of the total RNA is amplified. The tissue type, its developmental state, and its health all influence the actual proportion of mRNA in a total RNA sample. Total RNA from brain, testes, and embryonic tissues may contain up to 4% mRNA, while RNA from many other tissues will have only 1% or less mRNA. The RNA isolation method can also influence mRNA content. The generally accepted average value for mRNA content is about 2% of a total RNA sample. When 1 µg of total RNA, 2% or 20 ng of which is mRNA, is amplified 1000-fold, yields of 20 µg aRNA (or cRNA) should be expected. You may observe higher fold amplification when starting with lower amounts of total RNA. This is because, in an in vitro transcription (IVT) reaction, a finite amount of RNA can be synthesized with the fixed amount of NTPs. When starting with less RNA, NTPs do not become limiting until the RNA is amplified beyond the typical 1000-2000 fold amplification levels seen with higher amounts of input RNA.

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First, optimize the visualization of the image in the live video at 2X by increasing or decreasing the brightness setting, then right-click on the overview image and select "remember settings". This resets your optimized image settings, and when you select "re-acquire overview image" you will now generate a perfectly exposed overview image.

For a few cells: The gentle IR-only approach is the best recommendation for preserving nucleic acids and verifying that the desired material is collected on the cap. For groups of cells or large tumor regions: Mount your tissue on a membrane slide and take advantage of the power of the IR, or IR + UV cutting laser to cut around your region of interest quickly and cleanly.