2 replies to this topic

[hianghao]

I've extracted mosquito total RNA using RNeasy Kit from Qiagen. When i run it in MOPS formaldehyde gel, it showed 1 band! For ~20 samples that i've extracted, all showed single band of ~2000bp! Anyone knows what happen?
This photo shows RNA extracted in 1st + 3rd lane. 2nd lane is the control human total RNA. It seems like my RNA don't have 28s RNA!!

Edited by hianghao, 14 March 2011 - 12:32 AM.

[LabLackey]

My guess would be RNase contamination.  Are you using an RNase inhibitor (i.e. RNase Zap) to 'disinfect' all of your working surfaces, including gel box, tray, and lid?  Also, it looks like you have (slight) smearing on your samples, which leads me to believe that either they weren't boiled long/hot enough, or that indeed you have RNA degradation going on.

I don't work with mosquitoes, but do you use a mortar and pestle to grind them up?  Are you disinfecting those too?

[hianghao]

LabLackey, on 14 March 2011 - 01:40 PM, said:

My guess would be RNase contamination.  Are you using an RNase inhibitor (i.e. RNase Zap) to 'disinfect' all of your working surfaces, including gel box, tray, and lid?  Also, it looks like you have (slight) smearing on your samples, which leads me to believe that either they weren't boiled long/hot enough, or that indeed you have RNA degradation going on.

I don't work with mosquitoes, but do you use a mortar and pestle to grind them up?  Are you disinfecting those too?

I've contacted some researchers that deal with mosquitoes and they confirmed that the 28s rRNA fragment in size similar to 18s. Thanks for your respond!