I have extracted RNA by Trizol method, and got good Nano drop results, (as can see in images), but when I run the Gel 1%, at 90-120 V, for 20-30 minutes, then got no bands, ( I have many times, and used fresh TAE water, and distilled the tank amd comb too, but after all tries got no bands,
But when I run the Qpcr by using reference Genes the Cdna (5 times diluted), then got 23-28 cq/ct values with 0.1-0.4 cq/ct errors,
So need your suggestions that RNA quality is Ok,?
Should I use that RNA/Cdna for further targeted Gene study?
Getting RNA bands are necessary for further study?
I cannot really help you out in qPCR, since I never had to use this method until now.
However, looking at your measurements, I can say that your RNA should be OK.
How much of RNA do you load on your gel in the wells?
Do you use any kind of loading dye that's mixed with your samples prior to loading?
How do you stain your gels?
Adrian Marc Hauri i have used 1.0 - 2.0 ul along with 1.0-2.0 ul Buffer,
i used the Gel stain ,
You could try what Ma Dejun is saying. I do not see the issue right now. What kind of stain did you use? Is it ethidium bromide?
Check the composition of your loading dye. It's recommended, loading for RNA should contain 95% formamide. It's help to separate RNA better.
Hi Hafiz Muhammad Rizwan ,
I recommend the protocol described in appendix C of Omniscript® Reverse Transcription Handbook (Qiagen). It works always. The load of One microgram of RNA is enough.
The ratios 260/280nm and 260/230nm are good indication of RNA contamination, but not for RNA integrity. RNA integrity is assesssed with RNA gel electrophoresis, bioanalyzer equipment or QubiT 4.0 equipment using a specific chemistry.
Important: RNA ribossomal bands must appear, otherwise you have a big RNA degradation problem.
Best,
Jacó
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