Hi!

RNA gel electrophoresisis checks 28S and 18S bands integrity. These bands give a measure of RNA integrity, usually 28S is twice strong than 18S band for good RNA.

Protostomates are exceptions, they have just one band because 28S rRNA has endogenous hidden breaks, which result in comigration of the 28S rRNA fragments with the 18S rRNA in denaturing gel electrophoresis (Ishikawa, 1977; Dheilly et al., 2011; Winnebeck et al., 2009).

260/280 measure protein contamination (1.8-2.0)

260/230 measure EDTA, phenol and carbohydrates contamination (2.0-2.2).

Try using low melt agarose and denaturing conditions, use the protocol contained in Appendix C of Omniscript® Reverse Transcription Handbook (Qiagen). Always works in my lab.

Dheilly,N.M et al. Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns. BMC Genomics, 2001. doi: 0.1186/1471-2164-12-468.

Ishikawa, H. Evolution of ribosomal RNA. Comp. Biochem. Physiol B., 1977. https://doi.org/10.1016/0305-0491(77)90116-X

Winnebeck, E.C. et al. Why does insect RNA look degraded? J. Insect Sci., 2010. doi: 10.1673/031.010.14119.

Best,

Jacó