Questions tagged [reverse-transcription]

Questions pertaining to the synthesis of DNA from RNA.

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Reverse protein biosynthesis in nature

This is more of a funny question to think about, but I am wondering, theoretically if it is possible in cells to synthesize nucleic acids(DNA, RNA) from proteins. What might this be useful for and how ...
bioHacker's user avatar
2 votes
1 answer

Why is cDNA usually lacking in terminal sequences of the template mRNA?

It appears from presentations I have attended that cDNA often lacks terminal sequences (usually 5′) which have not been copied from the mRNA. This puzzles me a lot, but I have not been able to find ...
Aurel's user avatar
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Are the controls for RT-PCR the same as those for RT-qPCR?

I am searching for negative and positive controls for RT-PCR but all the results seem to point towards RT-qPCR. Are the controls the same for both? I have found -RT control No template control ...
bohemian's user avatar
1 vote
2 answers

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 ? ...
Youloush's user avatar
  • 113
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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 ...
Jahnic Beck's user avatar
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Complementary DNA synthesis

Does the reverse transcriptase enzyme recognize thymine to synthesize the complementary strand of cDNA?
Ray Elizabeth Dominic's user avatar
3 votes
3 answers

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 ...
Ubdus Samad's user avatar
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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-...
uhoh's user avatar
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2 votes
1 answer

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 ...
Dan's user avatar
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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?
Sergio.pv's user avatar
  • 185
2 votes
2 answers

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?
Katz's user avatar
  • 1,013
4 votes
1 answer

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. ...
user137's user avatar
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3 votes
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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 ...
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1 vote
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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 ...
user avatar
9 votes
2 answers

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 ...
Earl's user avatar
  • 91
6 votes
2 answers

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 ...
kali281's user avatar
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