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RiboAmp® HS Plus
Complete Solution for Gene Expression Analysis
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1. Prepare Samples
Cut a 7 micron section from a frozen block. Stain and dehydrate tissue sections while keeping RNA intact.
2. Identify Cells of Interest
Isolate 10 to 500 pure cells from frozen tissue samples using the ArcturusXT™ or Veritas™ Laser Capture Microdissection Instrument or obtain tissue scrape from slide.
3. Extract and Isolate RNA
Recover high-quality total RNA using optimized extraction and isolation reagents.
4. Amplify RNA
Amplify as little as 100pg of total RNA from frozen tissue samples and generate enough aRNA for microarray analysis.
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5. Microarray Labeling
Optimized Labeling Kits from Molecular Devices offer a proprietary, non-enzymatic technology optimized for the labeling of unmodified, amplified RNA (aRNA) for gene expression profiling.
6. Profile Gene Expression
Generate gene expression data using various compatible microarrays, or Quantitative RT-PCR.
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The RiboAmp HS Plus RNA Amplification Kit allows researchers to perform microarray studies using total RNA from as few as ten cells. This ultra high-sensitivity kit is a hundred times more sensitive than other linear amplification methods.
RNA amplification from 10 laser-captured TM3 cells. TM3 cell cultures were cytospun and slides stained using Topro 3. Ten cells were Laser Capture Microdissected using CapSure™ HS LCM Caps with the PixCell® IIe LCM System and total RNA was isolated with the PicoPure™ RNA Isolation Kit. RNA was amplified for two rounds using the RiboAmp HS Plus RNA Amplification Kit. Approximately 300 ng of aRNA was analyzed by electrophoresis using the BioAnalyzer (Agilent Technologies) and aRNA sample profiles are shown. Red: 10 TM3 cells. Blue: 100 pg universal RNA.
RNA samples are amplified up to ten-million fold in two amplifications, enabling microarray hybridization experiments from as little as 100 picograms of total RNA, the approximate amount in ten cells.
With the RiboAmp HS Plus Kit, 100-500 pg of total RNA or as few as 10-50 cells captured by Laser Capture Microdissection (LCM) generate enough amplified RNA for cDNA or oligonucleotide microarray studies. For best results, use the PicoPure™ RNA Isolation Kit to optimize RNA recovery from small samples prior to amplification.
RNA amplification from 50 microdissected cells. Total cellular RNA was isolated from replicate microdissections of mouse cells, 50 each of liver and brain, using the PicoPure RNA Isolation Kit. Independent two-round amplifications were performed using the RiboAmp HS Plus RNA Amplification Kit, generating aRNA product of 200 to over 2000 nt. Amplified aRNA was electrophoresed on a 1.25 % agarose gel and stained with SYBR® Gold Nucleic Acid Gel Stain (Molecular Probes). M: Marker. Lane 1: Negative control without RNA input. Lanes 2-3: aRNA from 50 mouse liver cells. Lanes 4-5: aRNA from 50 mouse brain cells.
RNA amplification from 100 pg and 500 pg total RNA. Mouse TM3 total cellular RNA and Universal Human Reference RNA (Stratagene) were amplified two rounds using the RiboAmp HS Plus RNA Amplification Kit. Amplified aRNA was electrophoresed on a 1.25 % agarose gel and stained with SYBR® Gold Nucleic Acid Gel Stain (Molecular Probes). M: Marker. Lane 1: 100 pg TM3 cell RNA. Lanes 2-4: 100 pg universal human reference RNA. Lane 5-6: 500 pg universal human reference RNA. Lane 7: Negative control with no RNA input.
The RiboAmp HS Plus method maintains the original mRNA representation after two amplification rounds, enabling accurate gene expression profiles from ultra small samples. Comparison of microarray hybridization results between cDNA probes generated from total cellular RNA and from aRNA obtained after two rounds of amplification using the RiboAmp HS Plus Kit reveals very high concordance between differentially expressed genes. This demonstrates the amplification fidelity needed for histologically relevant differential gene expression data.
Expression between RNA amplified two rounds with the RiboAmp HS Plus Kit and total cellular RNA correlate well. Total cellular RNA and aRNA amplified two rounds using the RiboAmp HS Plus Kit (500 pg) were generated from the same pools of mouse testis and brain samples, labeled and hybridized onto a 15,000-element cDNA microarray. Differential gene expression ratios between brain and testis were calculated. High correlation (R = 0.89) between the amplified and unamplified samples indicates the high fidelity of the RiboAmp HS Plus Kit process.
When working with ultra small samples, amplification reproducibility between samples is particularly important. Data from multiple amplifications and hybridizations of the same starting material using the RiboAmp HS Plus RNA Kit correlate exceptionally well.
Microarray correlation between two independent amplifications. Independent two-round amplifications were performed on 500 pg total RNA from mouse testis using the RiboAmp HS Plus RNA Amplification Kit. Amplified aRNA was labeled with Cy5-dUTP during reverse transcription and hybridized to two 15000-element mouse cDNA arrays, a portion of which is shown. Hybridized arrays were scanned and fluorescent intensities analyzed. A Log2 scatter plot of relative fluorescent units (RFU) shows high correlation.
RiboAmp HS Plus Kit enables gene expression profiling from only 50-100 colon cancer cells. Replicate 50 to 100-cell samples from normal mouse colon and microadenomas from a mouse were Laser Capture Microdissected (LCM) with the PixCell® II LCM System. RNA was isolated using the PicoPure RNA Isolation Kit. Two-round amplifications were performed with the RiboAmp HS Plus RNA Amplification Kit, and aRNA was labeled with Cy3-dUTP and Cy5-dUTP during reverse transcription and hybridized to 15000-element mouse cDNA arrays. Panel A. Aberrant crypt foci from colon cancer tissue from mice before and after LCM. Panel B. Correlation of replicate normal arrays show high reproducibility (R = 0.98). Panel C. Several genes are up- and down-regulated between normal and microadenoma samples. Data provided by Drs. P.R. Nambiar and D.W. Rosenberg, Center for Molecular Medicine, University of Connecticut Health Center, Farmington, CT.
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