[cosmarium]

Agarose Gel image

Thanks for your help.

Attached is a picture of a gel scan (the ladders seem to be running differently but it is a pic/visual effect).

I think my problem may be that the input amount of RNA is too low. According to nanodrop measurement my RNA is 0.8 to 2.5 ng/uL. I know the protocol ask for a 1ug input, but I don't know how strict it is.

I think I am going to order primers to amplify my 150bp band to see if somethings is there in small amounts that gets amplified by a second PCR and then I can see it. This will allow me to know if the problem is too few input RNA with vast excess adapters, primers OR if my RNA amount is too small and is not being kinased properly, which would prevent adapter ligation.

It seems like my amounts are pretty small, however I have done experiments with RNA population sizes of 100 molecules in the test tube. That is yoctomol range, and after RT and PCR amplification I can see a DNA band in an agarose gel, so I don't see why the input amounts for this protocol have to be in micrograms, that is a whole lot of RNA!

My sample is a mix of random 24-mer oligonucleotides, and it is preferable to have small concentrations to start the experiments than large ones.


Many thanks in advance for your ideas,

C

[CuriousA]

Hey Cosmarium,

Did you ever measure your RNA content with qubit or sth. comparable (fluorescent dye binding specifically to RNA)?

In low amounts nanodrop can be very treacherous.


How many cycles did you PCR amplify your cDNA?

Perhaps it's worth a try to go up to 20 cycles or so and check if you then get the right band.

Best,

Anja

[cosmarium]

Anja, many thanks for your reply and I am sorry I never responded. I now work on a protocol for small nucleic acid concentration and seem to work fine.
All the best,
C

[robertami]

mouse smallRNA library Illumina Truseq

I thought I shared my results to see how they compare to yours or if anybody can reassure me I'm selecting the right product.
I made a smallRNA library from mouse tissue using the Truseq kit from Illumina. I gel-purified the band between 140 and 160 as recommended.
My only doubt is about the shape of the Bioanalyzer trace after gel purification, it looks much smoother and broader than the one shown in the Illumina protocol. Any of you has ever seen something similar? Might just be because it's mouse and not human samples?
thanks for any idea!