Questions tagged [reverse-transcription]

Questions pertaining to the synthesis of DNA from RNA.

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31 views

Non-radioactive nucleotide labeling compatible with reverse transcriptase?

Are there any methods for nucleotide labeling which are compatible with use for reverse-transcription, besides radiolabeling, and for which the regulatory framework in the EU / USA are permissive ? ...
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1answer
19 views

Minimum amount of RNA for cDNA conversion and qPCR

I searched several protocols for reverse transcription of RNA and all suggest using RNA concentrations of 1ug. Could a lower amount result in inferior transcript representation and therefore decreased ...
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0answers
28 views

Complementary DNA synthesis

Does the reverse transcriptase enzyme recognize thymine to synthesize the complementary strand of cDNA?
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3answers
1k views

Why do retroviruses go through a DNA stage to replicate their RNA genome?

Why do retroviruses (e.g.HIV) convert their RNA genome to DNA (using reverse transcriptase) and then transcribe it back into viral RNA (and translate that into viral proteins). Surely to replicate ...
1
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1answer
74 views

How can they know that they found “all the PERVs”? (porcine endogenous retroviruses)

The New York Times article Gene Editing Spurs Hope for Transplanting Pig Organs Into Human links to the recent Science publication Inactivation of porcine endogenous retrovirus in pigs using CRISPR-...
2
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1answer
175 views

RT-qPCR for RNA quantification

I'm really new to RT-qPCR and probably I've just a minor problem than I think. So, what's my problem about. I want to quantify a virus by a specific area within a gene on its genome. I've specific ...
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0answers
58 views

Same primers and different melting peaks for two samples (~1°C appart)

In the figure, you can see that melting peaks differ by ~1°C at each sample. Should I be worried?
2
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2answers
58 views

Measuring reverse transcription yield?

Is there any measurement I could perform after RT that allows me to check the efficiency that the procedure had? Nanodrop cannot be used as remnants of RNA and poly T primers mask measure, or?
4
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1answer
191 views

Large scale reverse transcription?

I need to make RNA:DNA duplexes. I can make 100 to 200 ug of mRNA through in vitro transcription, and I know how to use reverse transcription to make a cDNA library, but I have questions with this. ...
3
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1answer
44 views

HIV-RT nucleotide contact residues

I'm investigating HIV-RT 3KLF and 1LWF trying to find a perfect pose of thymine. Which residues on HIV-RT is Thymine supposed to contact on 3KLF and 1LWF? Where exactly is the nucleotide binding ...
1
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1answer
111 views

qrt-pcr and short fragments

I plan to measure the effects of Tenofovir a Nucleotide analog reverse-transcriptase inhibitors useing qrt-pcr with HIV-RT as the rt enzyme. Tenofovir causes early termination of the reverse ...
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2answers
144 views

Is there a Reverse Transcription optimization for long, 9kb, transcripts?

Has anyone optimized RT for long transcripts (9kb)? The downstream application will be PCR amplification and Illumina library prep. It will be trivial to make internal primers sets for the PCR that ...
6
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2answers
1k views

Reverse transcription PCR optimization

What is the ideal amount of RNA to use for the RT? and how much cDNA to use then for the PCR? I did RT with a solution of RNA of 0.36 ug/ul. Then for my PCR I used 1 ul of the cDNA obtained and used ...