I want to do DNA micro-array for MRSA treated with compounds to study the gene expression level, during isolation of RNA using different kit methods ( Qiagen, thermo) concentration of RNA in nanodrop is a very good concentration and purity, but under the gel it look like it's smearing. I used all reagents which is very rnase , and treated by DEPC H2O. Is there any effect of Gel, or TBE Buffer for searing?
It seems you RNA is degraded. this can be detected only on gel not by nanodrop . If you suspect you can recheck by denaturing gel also.
RNA degradation also occurs due to endogenous RNAses so it is recommended to select such a RNA purification protocol that removes RNAses in the initial stage, such as add 3 ul of proteinase K to the buffer in which the sample is initially homogenised. Moreover high voltage and heat during electrophoresis also degrades the intact RNA so try to run the non-denaturing gel at 55 V for 2 hours. If u are using formaldehyde RNA denaturing electrophoretic system, then the formaldehyde pH must not be below 4 as it also degrades intact RNA.
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