I want to first amplify the HIV-1 env gene from a patient's plasma samples and then I want to clone it into a plasmid vector. I am using the QIAamp viral RNA mini kit for the extraction of HIV-1 RNA from the plasma. I have extracted RNA from a 560 µL plasma specimen. The problem is I have failed to generate cDNA and subsequently failed to amplify the env gene. Viral load in the serum ranges from 4000 to 550000 copies /mL. For cDNA synthesis I have used ReverTra A reverse transcriptase.
I would appreciate it if fellow researchers would share their experiences with plasma samples from HIV-1 positive patients.
Dear colleagues,
Thank you very much for your responses. Finally I followed the CHAVI protocol from UAB. It worked. I used the same primers mentioned by them. Finally, I think the problem was in primers. Now I am using the primers mentioned by CHAVI protocol.
Special thanks to Martin.
University of Alabama has a nice protocol for Single Genome Amplification of Envelope. The cDNA synthesis step is included: http://www.uab.edu/cfar/images/chavi-mbsc-14.pdf
I don't know about ReverTra A reverse transcriptase, but I use Superscript III Reverse Transcriptase and it works fine.
I use standard protocol and kit from Strategene, and it was working fine.
Dear Ridolfi,
Thanks for your answer. I am using Oligo dt (20). For positive control I am using JRFL clone plasmids. PCR is working fine for it. The problem is clinical samples.
Hi Muntasir,
You can use Titan one tube RT-PCR system from Roche (https://cssportal.roche.com/LFR_PublicDocs/ras/11888382001_en_09.pdf) with a set of outer primers and then use expand long template pcr system from Roche with a set of inner primers (nested PCR). purify and its ready to clone. Expand long will produce blunt end pcr products and also with A oferhangs, so it will suit your vector.
Clinical samples are alway harder. You need to optimize it differently. Good luck :)
Thanks Francisco. Personally I don't like Roche product. They keep lots of information confidential and while working with clinical sample or your own experiment setup sometimes you reach in a dead end with Roche.
Dear Barbara, I noticed that I have not treated my cDNAs with RNaseH, can it be a problem? What do you think.?
Dear Muntasir,
Attached is SOP that might help, i've based my experiments on it before. I've allready used more than 2 diferent Kits to produce cDNA "transcriptor high fidelity cDNA synthesis Kit" its rubish takes ages and almost impratical arround here, i was using the primers from the Kit (random hexamer primer) and i couldn't produce cDNA. The one i've talked before its good and fast, and know i use my own set of primers. 4000 copies/ml isn't much, when i have such a litle amont i concentrate first (< 100.000 copies/ml).
Hi,
there are some good links and suggestions mentioned above. I will add one more consider. As I remember HIV-1 and env gene has a GC rich content, in some cases in can influence on the cDNA transcription and further DNA amplification. Just keep in mind on that. There are some good RT and PCR buffers from NEB and Qiagen to avoid these troubles.
Good luck!
Dear colleagues,
Thank you very much for your responses. Finally I followed the CHAVI protocol from UAB. It worked. I used the same primers mentioned by them. Finally, I think the problem was in primers. Now I am using the primers mentioned by CHAVI protocol.
Special thanks to Martin.
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