cDNA synthesis

[ifhmn]

I'm going to do my RT-PCR. but before that I have to synthesis my cDNA. The problem is that I have a low concentration of RNA. For example, I have a concentration of 44.3ng/ul in 35 ul of sample (S1, so total yield of RNA is 1.5ug and 31.5ng/ul in 35ul of sample (S2) so, total yield of RNA is 1.1ug. Both S1 and S2 are from the same samples. THen, the protocol from the bioline (from cDNA synthesis kit) used 1ug of RNA in a total volume of 10ul of DEPC water. Can someone help me on how to synthesis my cDNA.. Can I mix S1 with S2 so that I can have a more high yield of RNA??

[luisillo]

If the samples have the same origin then there should be no problem if you mix them. However, in your case I don't think that would help you, because your RNA conc will increase but the volume would be still too much for RT reaction.

The only way I see to someway overcome your issue is to precipitate the RNA again and resuspend it in a smaller volume of DEPC water. But be aware that this could lower your RNA yield a bit.

[ifhmn]

thanks. Before this I resuspend my RNA in 40ul of DEPC. Is it ok if I resuspend it in 10ul of DEPC or it is too little?

[admin]

IFHMN,
are you using DEPC or DEPC treated autoclaved water?

[luisillo]

Yes, as long as you can completely dilute the RNA and you have enough volume to both quantify it and run the RT.

[ifhmn]

I used DEPC treated aotuclaved water. Actually, how can I completely dilute my RNA? just pipette it up and down or should I vortex it?

[luisillo]

I don't think vortexing would be that effective since the volume will be too small (10 ul) for effective mixing. Then I'd suggest you solve it by pipetting.

[ifhmn]

I have synthesized my cDNA, did the PCR reaction and run the gel, unfortunately, no band appeared accept for my housekeeping gene (ubiquitin). I used 1ug and 2ug of RNA to synthesize my cDNA. I dont know what is wrong. I have also tried using DNA to check whether my primer is working or not. The good news is that, I got bands for my PCR product using DNA sample. And then I try repeat again today using the same sample of my DNA and I ran the gel together with my PCR product from cDNA. suprisingly, no bands appeared for both samples (PCR products from DNA and cDNA). Does anyone know why this was happen???

[luisillo]

About cDNA, not having bands in PCR either means you have no or not enough cDNA, or you have too much. I'd go with the first considering your past posts, but you could use the nanodrop to know how much cDNA you have.

About the PCR not working again with DNA, I think it may be the DNA. It might be degraded or something, so I recommend you try using a different one.

Don't get stressed, these things happen often.