HIV RT gets processed by an E. coli protease during expression to make the RT heterodimer, but I do not know how good it works in an RT assay. I did express an RT in insect cells, and then purified it, takes some work! You could look for papers on MMLV and AMV RTs expression/purification and then get the gene synthesized and express as described in the papers.

Thanks Gary. Many years ago HIV RT was being "given away" as part of the HIV AIDS directed programmes. It was used to test the effect of/ screen for RT inhibitors so I presume it must have been reasonably active but don't know in the context of real-time RT PCR. Which RT did you express - did it require glycosylation for activity ? I did look up some papers -quite old now.

It was the RTBV RT, was not glycosylated, it was tested by a scientist at Invitrogen but it was not as good as AMV RT (I think) in terms of "in vitro" cDNA extension length, that is what MMLV and AMV RTs are good for.

Eons ago a friend of mine did it with AMV RT. My recollection is that it was a long, labor-intensive process (there wasn't much disposable labware available at the time), and none of his batches could compete with commercially available AMV-RT at the time (this was in the early nineties). Not cost-effective, if you ask me.

I am a little surprised that with His Tagged protein technology, TEV cleavage , FPLC etc which was not readily available when I used to purify enzymes, that the process is not more straightforward. For most RT-PCR where the target amplified is

Hi!

I prefer to buy the Promega products. not the kits themselves, but the enzyme (w/buffer), RNase inhibitor and oligos dT separatelly. We find the Altenzyme Taq and dnTPs mix much cheaper than other brands. I I can tell you, Our lab works with very tight budget!

Hi Julio,

Could you please tell me how you get the Promega products ( Separated one).

Cheers,

Issa

I agree with Julio, the use of Promega products or other authorized companies will be better, so I think if you contact the company and they will advice you, you can find they contact telephones or emails in their website.

Best Luck

Hi,

Consider the aim of your RT is for quantification, trying to use a home-made RT for saving some reagent cost. may not be the best option. The accuracy and repeat ability of the quantification are much more important . I used to make my master mix for RT and qPCR. However, I strongly recommend to use reputable commercial kit. The late data analysis and result comparison with other lab are much easier.

Thank you to those who have contributed. I am fully aware of the variety of kits available and the necessity for QC and have tried a number of kits over the years having done PCR since the mid 80s.. The point of raising this issue is try and see whether it is possible to get the cost of RT to less than $1-$2 per reaction - this would enable a greater number of labs in resource poor countries to undertake the sort of analysis that is available to well funded labs. Often the places where diagnostic resources are most needed are the places which have the poorest access to technology because of the cost. If your health budget is tens of dollars per person per year as opposed to several thousand then you will not be able to afford this technology. The cost of RT has in our experience risen over the past few years. This is not a brand new technology, and I would have expected the cost of recombinant protein to have actually decreased with time as it has for most restriction enzymes, Taq etc. The query about making RT was to try and discover why it is so difficult to make RT and if the price is justified or just a reflection of our willingness to accept the status- quo. Most people just accept whatever the suppliers tell them.

Hi Ian Teo,

I agree with you and I am looking forward to hear from you about an approach to homemade RT. If you need human resource to develop futher this your idead, you can count me in it, ok!?

Cheers

Luís

As mentioned by contributors above, issue of badge-to-badge efficiency, stability and reproducibility of the data matters for quantification purpose. You may further cost down by using half of the suggested volume (such as 0.5 ul per rxn instead of 1 ul). It works fine in our lab using Promega RT and other generic brands of RT which are much more cheaper. You may ask for trial samples for testing.

I have been using RT kit or gel pellet from Bioneer of Korea, it is relatively cheaper and can be customised to include with primers for high throughput usage.

I agree with Julio, the use of Promega products will be better

Sorry for the delay. The answer to the Issa's question. I checked the Promega internet page and order the products separately by their catalogue number. For example the recombinant Rnasin ribonuclease inhibitor has the part. no. N2515. I looked for it in the www.promega.com page and select the product I need. Some times the product numbe in the kit is different from the product number that is sold separately. but just be careful. Promega and other companies sold many of their kit parts, separately.

good luck!

I totally agree with Julio and Mostafa, promega was a good company and you can found what you need online, good luck for you!

I use Promega MMLV and RNAse inhibitor, dNTPs from any well known producer, and my RT does well...

the key is to check the efficiency e.g. with RT-q PCR for ABL transcript... and see how many copies you obtain with the commercial kit vs. your in house procedure... then you can choose. Good luck !!

I'm currently considering trying for home made RT. I found the following paper (http://link.springer.com/article/10.1007/s10529-009-9977-5/fulltext.html) for a potential protocol which I consider trying.

We have been using our own RT reaction setup for many years with great success (

Hello Robert. I have been using various concoctions of RT kits with add ons - Arctic shrimp DNAse, RNAsin etc. for a number of years.  In all my RT work, we avoided RT mixes that had DTT as a stabilizer as it had a deleterious effect on the subsequent PCR - we showed this by spiking increasing amts of DTT or mercaptoethanol into PCR mixes and seeing a drop offf in product generated.

There are also other issues of inhibition of PCR by RT (see below).

We had contemplated reducing RT to half that recommended but never got  round to it.Reaction times are also usually excessive - there is no obvious reason why reactions are 1h, when in reality they can be reduced to 15min +

So far no one has really come up with an explanation for why the cost of RT itself is so exhorbitant and has increased over the years, which is the issue I was hoping to address rather than the merits of different kits /mixes.

See references in

http://nar.oxfordjournals.org/content/33/20/e181.full

PCR inhibition by reverse transcriptase leads to an over estimation of amplification efficiency.

• Oleg Suslov and 
• Dennis A. Steindler*

You can find a nice protocol and discussion about different RT available here: https://pipettejockey.com/2018/09/06/mashup-rt-purify-your-own-reverse-transcriptase-beta-testing-phase/