Dear DavidGene expression is measured by the amount of mRNA therefore it must be extracted from the sample cells or tissues. In some cases total RNA can be used. It is very important to have high quality RNA (i.e. the sample has a low percentage of degraded RNA), as without it the gene expression results will be meaningless, so it is important to check the quality before proceeding to apply the material to the microarray. The quality of the RNA can be checked by using standard techniques such as agarose gel electrophoresis. More recently though this analysis has been greatly improved by the introduction of micro-capillary
based devices (for example the Agilent Bioanalyser) which requires a smaller amount of RNA and provides accurate quantitative results. As about 25-100 µg of total RNA is required in many cases the RNA needs to be amplified. In this amplification step it is important that every diffierent mRNA is amplified by the same amount or the gene expression results become meaningless. This is difficult to do but there are techniques that allow amplification in a linear rather than exponential manner which suit this task. For high density microarrays
one has to convert mRNA into complimentary RNA (cRNA) because the oligonucleotides sequence are in the sense direction and so one has to use antisense RNA which is cRNA. For spotted arrays one can use either mRNA, cDNA or cRNA because both strands are used as probes on the microarray.
Dr. Vikas Sonakya
Post Doctoral Fellow
Division of Biotechnology
Department of Physics and Measurement Technology
Linköping University
S - 581 83 Linköping
Sweden
vikso@ifm.liu.se
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RE: cRNA vs cDNA,
vikassonakya, Nov-03-03, (1)