RNA extraction Kit vs Trizol

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
c1bl
Garter
Garter
Posts: 8
Joined: Fri Jul 13, 2007 9:36 pm

RNA extraction Kit vs Trizol

Post by c1bl » Fri Jul 13, 2007 9:47 pm

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.

cheers,
Chris
Attachments
gel10 copy.jpg
RNA gel

gothica
Garter
Garter
Posts: 2
Joined: Sat Jul 14, 2007 12:27 pm

Post by gothica » Sat Jul 14, 2007 12:48 pm

Hi,
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.

MSc student
METU (Turkey)

gothica
Garter
Garter
Posts: 2
Joined: Sat Jul 14, 2007 12:27 pm

Post by gothica » Sat Jul 14, 2007 12:52 pm

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.

MSc student
METU (Turkey)

c1bl
Garter
Garter
Posts: 8
Joined: Fri Jul 13, 2007 9:36 pm

Post by c1bl » Sat Jul 14, 2007 1:16 pm

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)?
Attachments
gel10 cop2.jpg
I adjusted the brightness so you can see the faint bands better in the Trizol lanes 3 and 6. Could this be contamination of some sorts?

sdekivit
King Cobra
King Cobra
Posts: 586
Joined: Sat Jul 30, 2005 7:16 pm
Location: holland
Contact:

Post by sdekivit » Sat Jul 14, 2007 6:47 pm

what are the specifications of the kit. Maybe you should compare the experimental details of both experiments to descide what is going on here.

c1bl
Garter
Garter
Posts: 8
Joined: Fri Jul 13, 2007 9:36 pm

Post by c1bl » Sat Jul 14, 2007 9:32 pm

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?

sdekivit
King Cobra
King Cobra
Posts: 586
Joined: Sat Jul 30, 2005 7:16 pm
Location: holland
Contact:

Post by sdekivit » Sun Jul 15, 2007 6:14 am

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.

ozzmosis
Garter
Garter
Posts: 1
Joined: Fri May 30, 2008 10:45 am

Re: RNA extraction Kit vs Trizol

Post by ozzmosis » Fri May 30, 2008 10:57 am

uh, why is the gel picture upside-down? is it intentionally?

Post Reply

Who is online

Users browsing this forum: Bing [Bot] and 10 guests