dear Martinho, i am confused, whether your cDNA is ok or not?

if it gives perfect bands with other primers (specially house keeping primers) than it may be partial degradation of your cDNA b'coz of any contamination. your gene of interest may be in low amount and a slight degradation may do so. so plz do a pcr with freshly prepared cDNA from freshly isolated RNA of a good quality,.

i have fought with such a problem for a long time...

wishess

well yes i faced the same porblem but was successful in changing number of cycles to 30 and also yes i agree with Sheikh Asraf.. RNA quality and quantity must be checked.. for sure cos when i repeated three times... rna quality was not good that i was neglecting...but this should be taken care of

Dear All,

A similar problem was faced by us recently trying to amplify a HDAC6 gene from Total RNA sample. We tried using various types of MMLV RT enzymes from different brands for cDNA synthesis. We kept Actin gene as a control. It amplified each time yet no amplification for HDAC6. We used single step thermus thermophilus polymerase RT PCR. The same occured again. The result was mysteriously strange. Had there been any problem with RNA, my control gene Actin would not amplified that well.

Even I was looking for suggestions on the way to proceed with the same.

To investigate the problem one approach may be walking the cDNA with primers say, the gene is 3.5Kb I would try out with primers which would amplify 2.5kb from somewhere in the middle of the sequence, similarly with 1.5 Kb and 1Kb. This will give me directions where in the cDNA the problem is?

If this thing works out well we can know that there might be some complication with the RNA structure. Possibly a secondary structure might be the reason. I know its rare but Reverse Transcriptases really cannot help this situation.

Looking ahead for your feed back on the same.

regards,

Gaurab

Are you sure your master mix, water as well as your gels are not contaminated because you are getting smear in control also?

Dear all,

Thanks a lot for all the replies. :)

Regarding the quality of my RNA/cDNA I was never considering degradation since I was able to detect actin and other transcripts from the exact same samples. Actually every single usual pcr is working for these samples except this one. However, as Raghuvansh and Shukla mentioned this smear can be a contamination of partial degradation that do not affect the amplification of more abundant things and affect the specific one since is very low.

This a complicated situation for 2 reasons:

1st - I 'm a bit stuck with this primers because this transcript is very similar to another one except for the 3' utr, so one of my primers has to be there to be able to be specific. The other primer I have has to be before this miRNA target site, since I'm testing this transcript in conditions where this miRNA is probably cleaving it.

2nd - the pcr was working before with the same conditions, I did not change anything, so indeed the more obvious problem is the cDNA or RNA quality. But now the pcr is not even working efficiently for the same samples.

3dr - this pcr was the only result i had, that was interesting for my PhD thesis. I'm a bit worried about this.

Anyway if anybody has experience with amplification of low abundant transcript I would be grateful. :)

Dear Gaurab Banerjee

I think your suggestion are good even for your problem. But, in addition, I would try to check if my RNA would have any contaminants, like phenol for instance. These will definitely inhibit your RT reaction. One solution for this would be to wash your RNA pellets with 100% etHO before using the 70% or other. This if you extract the RNA with trizol, otherwise I'm not experient with RNA Kits.

I use Superscript III and is very good. For long transcripts you can increase a lot the extension (3h) time at 50C and it works quite good. Treating the cDNA with RNAseH is also very common solution for long transcripts.

I hope you solve your problems.

Cláudia

Thanks a lot for the reply. To your question regarding extention time and annealing temperature I had already modified my PCR Protocol annealing at 58C then we lowered it further to 55C, everthing was tried, extention time was the same as mentioned by you. We have tried a lot of permutations and combinations to get it fixed but failed.

We used high fidelity Taq and high Performance MMLV RT. SuperScript III was not tried but at least some result is expected. We did not go for any other extraction protocol as we know that spin columns reduces yeilds. Trizol method was uptaken.

Well I think I would have to try out working with other housekeeping genes to be hundred percent sure.

With regards,

Gaurab Banerjee