Drosophila Northern Blot and RNA questions - (Jun/26/2009 )

Hi, y'all,

I did Northern by the protocol of Escrich 2001 Biotechnology Letters, and detected the signals by NBT/BCIP, several questions:

1. This protocol ask for up to 20 ug total RNA to load, which is for mammalian tissues, I read some drosophila paper ask for 10ug and I did once it's okay for my control GAPDH2 (But when I stripe the membrane I lost everything...opps). So can I reduce it more to 5ug total RNA. My bands for 10ug was strong bands and looks good.

2. I use RNA from Drosophila early 3rd instar larval CNS. I can only get about 1-2 ug total RNA from 30 CNS tissue. Is that right or I had a low yield? I used trizol to extract.

3. I crosslink the RNA to nylon membrane by UV illuminator (the one we check DNA or RNA gel), 1min 30s for each side. Is that right?

4. Can I keep the membrane -80 degree for a while then do the hybridization another day? My PI said after you crosslink it everything may be fine. So at what degree should I keep the membrane and how (in some buffer) and how long and I leave it there?

Thanks. I just have to redo these and hopefully this time it will work.

Tina

Tintin on Jun 26 2009, 01:35 PM said:

Hi Tina-
1. This depends entirely on the abundance of the mRNA you are trying to detect. Highly expressed genes like GAPDH are easily detected from 5 ug total RNA or even less while other genes with low expression level can be difficult (sometimes impossible) to detect even from 20 ug total RNA. I think you should use as much RNA as you can especially if you are not doing radioactive labeling. You would be sorry (sorrier) if you use little RNA and fail to detect anything.

2. Drosophila tissues are so tiny so 1-2 ug sounds pretty good to me. Trizol prep tends to have contaminating DNA so the actual amount of RNA may be less than you think.

3. I haven't done UV crosslinking for a long time so I don't remember how long it should be (it also depends on the strength of UV, I'm sure). But I don't think you need to illuminate on both sides. Just do it on the RNA side (the side that was making contact with the gel).

4. I believe you can store the blots for at least a few weeks at room temperature. I used to store my unused Southern blots wrapped loosely in aluminum foils at room temp and they were good for many months. As your PI says, once crosslinked and kept dry, RNA as well as DNA is very stable.

Thank you so much, namekuji.

Room temperature to keep the RNA membrane? If I keep them in -80, does that hurt?