- July 13, 2007 at 9:47 pm #7952
Lane 2 and 3 are from the same tissue sample. 7mg of tissue used for each sample. Lane 2 is using a RNA extraction kit using filters and Lane 3 is using the Trizol method. Lane 4 and 5 are also the Kit and lane 6 is Trizol. Do I have DNA contanmination in my Trizol lanes? Why does the kit pick up the first band and the Trizol doesn’t.
- July 14, 2007 at 12:48 pm #74567gothicaParticipant
Actuallay if it is a mamalian sample we should see 3 RNA bands (rRNA bands) if your sample is taken from plant we expect to see four bands. In your gel we saw three bands isolated by kit and 2 bands isolated by trizol. To me it isn’t DNA contamination. If it was DNA it had to run slower than RNA samples and we had to see band at the back of RNA bands. The possible reasons:
Degredation of your RNA sample. So you have an additional band while using kit. Secondly RNA samples may form secondary structure. To eliminate this possibility you can treat samples at 72 C for ^2 minutes, or you can mix formaldehyde and formamide and put into gel when you prepare gel.
- July 14, 2007 at 12:52 pm #74570gothicaParticipant
Uupss, I am so sorry. I couldn’t realize the application site. Now I change my comments. It should be DNA contamination. You can apply DNase tratment and then run again. I had same problem one times and ı repeated DNase treatment step only. It worked.
- July 14, 2007 at 1:16 pm #74571
My Samples are from the leaves of aquatic plants. Each sample was taken from ~7-10mg of tissue. Application site is where the lanes are labelled 1-7. Also the Trizol method shows more faint bands towards the end of the gel. The kit uses glass fiber matrix columns to filter out the RNA. It says it effectively isolates RNA >200bps. Would the Trizol mehtod be more sensitive at getting lower MolecularWeight RNA than the kit (Lane 3 and 6)?
- July 14, 2007 at 6:47 pm #74576sdekivitParticipant
what are the specifications of the kit. Maybe you should compare the experimental details of both experiments to descide what is going on here.
- July 14, 2007 at 9:32 pm #74585
As for the experimental details. Lane 2 and 3 are Identical the only variable I changed was the method of extraction Kit vs Trizol. I have heard that the kit isn’t as good at isolating the Small RNa’s. I’ll eventually be running RT-PCR to determine genes that are differentially expressed during development of my plant. Most genes which are differentially expressed are rather small are they not? So should I be using the Trizol method to extract my RNA instead of the kit?
- July 15, 2007 at 6:14 am #74596sdekivitParticipant
well as you heard that the trizol method (which is used very often as far as i know) can also isolate small RNA molecules and the kit doesn’t do it as good, I would go for the Trizol method but it depends on what fragment you are interested in.
–> in your figures by the way, the RNA bands representing larger RNA fragment are missing so your statement may be true.
- May 30, 2008 at 10:57 am #84354ozzmosisParticipant
uh, why is the gel picture upside-down? is it intentionally?
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