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How much total RNA is needed for RNA Amplification? |
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Recommended input amounts of RNA are 50ng Fresh Frozen total RNA and 100ng FFPE total RNA. Up to 200ng RNA may be used. |
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What kit/method should I use to isolate my RNA? |
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The following kits may be used for isolation of FFPE total RNA:
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RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Applied Biosystems/Ambion)
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Agencourt FormaPure Kit (Beckman Coulter Genomics)
Fresh Frozen RNA may be isolated with any kit or technique previously shown to be compatible with microarrays. |
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Should I treat my RNA sample with DNAse? |
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DNase treatment of the RNA sample is recommended, as contaminating genomic DNA will also be amplified by Sensation. After treatment, completely inactivate the DNase by the recommended method, heating, or phenol-chloroform extraction. |
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What are the most critical points in the procedure? |
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cDNA Synthesis: Do not use expired reagents.
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Purification: Follow the Genisphere protocol for purification.
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Promoter synthesis: The tailing step should not exceed 2 minutes.
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IVT: Thaw the T7 Nucleotide Mix and 10X T7 Reaction Buffer at room temperature, and keep at room temperature until use. Thoroughly vortex the 10X T7 Reaction Buffer to resuspend. Prepare the IVT Master Mix at room temperature, keep at room temperature, and add to samples at room temperature.
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What is the typical fold-amplification? |
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The fold-amplification depends on the quality of the input RNA sample. Typically, at least 10ug senseRNA should be obtained. |
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Will the use of random primers in the first step, cDNA Synthesis, amplify ribosomal RNA in my sample? |
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Yes, Ribosomal RNA will be amplified, and does not affect microarray results when appropriate stringency is maintained. Specificity is defined by the array itself, using the recommended hybridization conditions. |
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How degraded can an RNA sample be, for use with the Genisphere kit? |
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RNA samples with RIN values of at least 2 can be used with the Genisphere kit. |
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What are the specifications for total RNA on the Agilent Bioanalyzer? |
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Currently Genisphere does not have any specifications for total RNA on the Agilent Bioanalyzer. |
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Can I amplify microRNA with Sensation? |
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No. Please contact Genisphere Technical Support if you would like to amplify microRNA. |
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How can I test reactions in the kit? |
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The Control DNA (Yellow cap) can be used to test reactions after First Strand cDNA Synthesis and Purification. Follow the procedure in the Appendix for troubleshooting purposes, to ensure kit components are working appropriately. |
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How do I contact Genisphere Technical Support? |
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Toll Free: 877.888.3362 or email techsupport@genisphere.com |
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