RNA extraction

[rp_srk]

hi!

I am a new memeber to this forum. I am doing PhD in molecular bilogy. My work is to generate transgenic tobacco plants and to analyse the transcript levels of the transgene at different stages of leaf development. For this, I took very young, mature and the very old leaf as senescent leaf. I extracted RNA from all the leaf samples using Trisure. But the problem is that the RNA is getting degraded in the senescent leaf samples and mature leaf samples. Initially I thought it was a problem in handling the material but even the second time extraction remains the same. Hence I am unable to decide the reason for this. I would be very thankful if some one explains me the reason for this - if it is due to handling or in plant cells itself the RNA degrades faster in mature and old leaf samples.

 

Thank you,

 

[Bluenoise]

Well if the RNA is getting degraded I'd automatically think of RNase. The only explaination that I can offer for why your very young leaf is working is because it likely has a higher level of RNA in it. Faster growing cells need more RNA than stationary cells.

So are you sure that you're redisolving in DEPC treated water, and everything is very RNase free?

Another possibility is that the RNA is getting degraded before the Trisure is able to deactivate any RNase present. I'd recomend pulverising your leaf into a powder with liquid nitrogen prior to the addition of the trisure.

However you may already be doing this. If so I don't really have an idea.

[ecoli]

RNA work is tricky because it is very susceptible to degradation. Make sure all you reagents are RNase free.

 

Also, you may want to add in a reverse transcription step, because DNA is easier to work with than RNA.

 

At least, that's what I did when measuring gene expression levels in e. coli.

[CharonY]

Depending on what he wants to compare this is not an option, as RT can induce a bias in the level of detection (so basically it is a matter of normalization options). It might be easier if a protocol is posted.

[ecoli]

Depending on what he wants to compare this is not an option, as RT can induce a bias in the level of detection (so basically it is a matter of normalization options). It might be easier if a protocol is posted.

 

generally, you can use a housekeeping gene to do this, one where the expression level is fairly constant as compared to total expression.

[Bluenoise]

generally, you can use a housekeeping gene to do this, one where the expression level is fairly constant as compared to total expression.

 

He means that the process of reverse transcription doesn't treat every transcript equally. IE some are over represented and vise-versa when converted into DNA.

Use of a gene of constant expression can not help with this problem.

[ecoli]

He means that the process of reverse transcription doesn't treat every transcript equally. IE some are over represented and vise-versa when converted into DNA.

Use of a gene of constant expression can not help with this problem.

oh, I did not know that. Do you happen to know why that's true?

[Bluenoise]

I'm not 100% positive, as this isn't a technique I typically work with. However, It likley has to do with primer bias during the reverse transcription step.

[CharonY]

In part,yes. Of course for eukaryotes you'll use a poly-T primer, in that case the primer bias is lower. Yet, efficiency of RT is also dependent on the template itself. Secondary structures can be a problem, for example. Also your RNA may degrade during your RT reaction, so that certain instable low abundant RNAs are getting lost, introducing yet another bias. Despite all that for certain techniques (e.g. microarrays as well qPCR) you have to make an RT step, though. It is only important to keep in mind that a bias is possible (and even likely).

 

Regarding housekeeping genes: afaik there are hardly genes that fit the bill. This is especially true for stress experiments.

[jcoalsonshore]

this tissue pulverizer gadget is useful for cell lysis in liquid nitrogen, especially fibrous stuff like plant material. The company is called Cellcrusher

 

cell lysis