( Frequently Asked Questions ...)RNA Amplification for Array Analysis

Frequently Asked Questions

A. How is fold amplification calculated?
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 embryoic 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 g of total RNA, 2% or 20 ng of which is mRNA, is amplified 1000-fold, yields of 20 g 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.
'"/>

Source:


Page: All 1 2 3 4
Related biology technology :

1. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Greater Amplification Specificity with New Hot Start PCR Enzyme
4. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
5. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
6. Improve Amplification Specificity with Hot Start PCR Enzyme
7. Amplification of the amoA Gene of Nitrosococcus oceani Bacteria Using Eppendorf MasterTaq
8. Amplification of Mouse cDNAs for Microarrays Using the Eppendorf MasterTaq Kit
9. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
10. Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler
11. Amplification of Epstein-Barr Virus Exon C with Difficult Primers Using Eppendorf Mastercycler gradient