RNA Extraction

[admin]

Hello,

I extracted total RNA from fish for the first time using QIAGEN RNeasy Kit. Well, when I checked my results, I got only 1 single sharp band and it situated somewhere middle of the agarose gel ( I run the electrophoresis for 1.5 hours at 36 V). This result was different from the standard.. with 2 bands ( 28s and 18 s ). Where I did wrong?
I have used native agarose gel ( TAE ) with 2x RNA loading buffer ( Fermentas ) for the electrophoresis. I incubated the RNA loading buffer with the RNA I obtained for 70 degree celcius for 10 minutes ( everything according to the protocol given by the supplier ). I have used an equal volume of loading buffer and my RNA sample during the incubation time.
I am confused now? Please if anyone could help me, it will be a very great for me.

p/s : should I use formaldehyde agarose gel ? or my exraction is wrong ?

Thank You so much !

Endran

[admin]

Hello Vljay!
welcome to the forums.

There could be several things that are going wrong. It seems the difference is the control RNA and your RNA. This could be because of the way Qiagen prepared the control. Are you using foraldehyde or heating your RNA?

You should be seeing the two bands (28S and 18S rRNAs) so I am not sure what step affected the running of your sample. I was going to say maybe you ran your gel improperly, however the control ran fine so I doubt it is your gel.

I have a feeling it is your RNA sample buffer and your sample preparation.

Did you make your own RNA sample loading buffer or did you purchase it?
How old is it?
Is there any streaking on the bottom of your gel?

Please report back here so we can help you more. If you find the solution, please post it.
Cheers!
Moleculardude

[admin]

Hi moleculardude,

I strongly believed that the sharp band that I got in my first RNA extraction was DNA and not RNA eventhough I didnt treat my sample with DNase.
So, I did my second RNA extraction using the Qiagen with some changes in the protocol and this time I got 2 bands. But again, this time I got a different ratio of 28s r RNA : 18s r RNA = 1: 2 and not the usual one .... 2: 1
Throughout my experiment, I am using RNA loading buffer ( 2x ) bought from Fermentas. I am preparing DEPC treated TAE buffer and I guess there is no problem in terms of Buffer prepration or gel prepration as I am getting a sharp, no smearing RNA marker bands ( RNA marker was bought from Promega).
The bands I got were approximately 3638 bp and 1908 bp when I compared with the marker ( RNA marker from Promega )

My question: is this RNA or some sort of DNA contamination ?


Please help me .

vijay

[deltafx1942]

it could be RNase contamination? RNase degraded your RNA on the first run for your sample, so you only showed one band, which you concede could be DNA. The fact that your Marker was fine suggests that it might not be RNase contamination of your procedure for the gel, just contamination of the preparation of RNA?

If you are not using formadehyde 1% gel agarose, then what are you using? I thought that was the only way to go. I did find a kit, the Elementary RNA gel kit from Amresco that doesn't need formaldehyde or ethidium bromide, but i don't know if that is the best way to go.