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Real-Time RT-PCR one-step and two step issue


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13 replies to this topic

#1 Curtis

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Posted 26 February 2009 - 09:19 AM

I'm trying to buy a SYBR Green RT-PCR kit for our RotorGene 6000 5 Plex HRM but I have some questions to ask.

for the moment I need to detect expression of mRNA in our cells so definitely I would prefer to buy a one-step kit, but the question is that in case I finish working with mRNA and then decide to work with DNA sample, can I still use the same one-step RT-PCR kit? because the first cDNA snthesis step shouldn't be interfering with my DNA samples. am I right?...if not....then wouldn't it be better to buy a two-step kit?

I still haven't decided to buy from which company and I am aware that company issues are not for this subforum, but still I would appreciate if you guys could let me know where you buy your kits from?

Edited by Curtis, 26 February 2009 - 09:21 AM.


#2 littleaxt

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Posted 27 February 2009 - 07:43 AM

In fact I don't know, because I never tried. But I ask myself, why do you want to spent more money than you have too? Why buying also RT-reagents when you don't need them?
Wow, wish I could affort that ... :-)

Good luck!

#3 Curtis

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Posted 27 February 2009 - 08:21 AM

it's not really my fault.....the kit is for 400 reactions....Qiagen doesn't have less than that. that's why....don't mock me:D

also Quantace supplier doesn't give less than 250-reaction kits. it's their policy....so I HAVE TO go for those kits altough I don't want to.

Edited by Curtis, 27 February 2009 - 08:23 AM.


#4 littleaxt

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Posted 02 March 2009 - 01:02 AM

Did not want to blame you :-)
And I better don't start talking about Qiagen....

I use Quantace and they also sell their SensiMix kit for 50reactions. You can downsize the reaction volume from 50Ál to 25Ál without any problem, so you would end up with 100 reactions. And you can order the same SYBR-green + Mastermix they use for their SensiMix seperately without the reverse trascription stuff.
But if you allready have bought the 400 reactions it's to late anyway.

All the best!

#5 Curtis

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Posted 02 March 2009 - 03:50 AM

still haven't bought. waiting for qiagen quotation to compare with quantace. thank you anyway.

#6 littleaxt

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Posted 02 March 2009 - 05:21 AM

ahh, sorry, you are doing HRM, that's propably an other kit (saturating dye...)

Good luck!

#7 NemomeN

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Posted 02 March 2009 - 09:37 AM

In theory, the answer to the original question is that yes you can use the cDNA for downstream applications from the one-step kits, BUT, you will need to clean it up as the buffers/salt does funky things for downstream applications (at least it's been my experience). I just ended up doing the whole thing with Trizol and then RT manually as I had more flexibility and consistency (plus it saved a ton of money too :rolleyes: Is the RT step a random hex/oligo dT or gene specific?

#8 Curtis

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Posted 05 March 2009 - 10:33 AM

hi,

thanks for your reply.

Qiagen kit has a reverse trascriptase enzyme called Omniscript which ,I think, there is no need to use Oligo dT with it anymore. this enzyme enables cDNA synthesis from viral RNA too. the kit is not gene specific, but I would prefer not to do Trizol extraction for real-time pcr.....is it really reliable to use trizol extracts in real-time pcr?

#9 NemomeN

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Posted 05 March 2009 - 02:18 PM

I've heard of omniscript, but have not used it, and I'm assuming if there's not oligo dT and it transcribes viral RNA then it's most likely random hexamers (or nonamers). Omniscript by itself assumes that you already have purified your RNA (unless it's part of a generalized kit that incorporates it into a one step process).

It's a question of practice. In the past, I had to use Trizol as the incubation step in one of the qiagen kits was too long and I got used to using it and with an extra acid phenol extraction, I've had good results. However, the one-steps are definitely easier, especially with small sample sizes.

But, in response to the original question which was whether the 1st strand cDNA could be used for other purposes after the qRT PCR, the aswer is yes, but you will need to purifiy it in some manner.

#10 jinx1987

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Posted 07 July 2009 - 10:50 AM

hi,

thanks for your reply.

Qiagen kit has a reverse trascriptase enzyme called Omniscript which ,I think, there is no need to use Oligo dT with it anymore. this enzyme enables cDNA synthesis from viral RNA too. the kit is not gene specific, but I would prefer not to do Trizol extraction for real-time pcr.....is it really reliable to use trizol extracts in real-time pcr?


I have used Trizol to extract RNA and protein simultaneously and then reverse transcribed with Invitrogen kit. The cDNA product works fine with real-time PCR.

#11 davidflora

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Posted 03 April 2012 - 02:58 AM

Hi all, I'd like to express snoRNAs (about 90nt) in mammalian cells and want to detect its expression by RT-PCR. I'm not sure can ordinary RT-PCR work for this purpose. Should I use random hexamer or gene-specific primer? I also plan to do northern blot in the future. Do I need to run acrylamide/urea gel or ordinary denatured agrose gel?
Thanks so much!

Edited by davidflora, 03 April 2012 - 02:58 AM.


#12 Trof

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Posted 03 April 2012 - 01:20 PM

In theory, amplicon in qPCR can be as small as 60-70bp, but question is if you can find suitable primers on such small area. I'm not sure about random hexamers, if they don't bind in your 90 nt at all, you won't get a product, so probably gene specific would be better. 2% agarose gel should have suitable resolution for 50 bp, but I'm not sure what bp difference it would be ale to distinguish.

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#13 muntasir

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Posted 08 April 2012 - 06:56 PM

I have used Qiagen one step PCR kit for PCR with DNA as template. Actually, the initial 95C step of conventional PCR will deactivate RT. In that case we can easily use one step RT PCR kit for amplification of DNA.

#14 Bassam27

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Posted 10 June 2012 - 10:44 AM

how can i extract RNA from blood quickly for RT PCR and RT LAMP ?




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