Reverse Transcriptase PCR

[Nathan McCorkle]

Josiah, I've tried using a few kits in the past to grab a transcript,
I was crunched with time though and only performed one experiment that
failed. I've since got two 3D printed bead beater attachments for the
Craftsman auto-hammer, the first time I tried using LN2 and a mortar
and pestle on a tissue sample. It was not easy, and I wasn't confident
it was sufficiently ground but I really couldn't tell. I actually used
a qScript qPCR kit, as well as some other RT one-pot reaction, both
with specific primers.

Anyway, what's your recommendation should I try this again?

On Tue, May 21, 2013 at 3:14 PM, Josiah Zayner <josiah...@gmail.com> wrote:
> People don't _not_ do Real Time PCR because the equipment is so expensive.
> They don't do Real Time PCR because it is not very reliable.
> Intra-experiment variability is very high. Protocols are complicated and all
> the reagents are expensive.
>
> With RNA quality being a hugely important factor most home labs are not
> equipped to do anything successful.
> Real Time PCR protocols that do more then detect copy number of a gene are
> complicated and require preventing DNA and RNAse contamination. You need to
> reverse transcribe your mRNA, chop up the DNA, chop up the RNA, purify the
> cDNA. And have multiple samples because your variability will be so high
> even on high-end machines much less a DIY machine.
>
> With Sequencing/Deep Sequencing starting to become really cheap and you get
> to see the copy number of every transcript not just the ones you PCR it is
> becoming the goto technique.
> The fact that one can just do Reverse Transcriptase PCR and run it on a gel
> if you want something quick and dirty makes Real Time PCR not a very good
> method.
>
> Real Time PCR is not a fancy technique that can do something nothing else
> can do.
>
> Most Real Time PCR used nowadays is for diagnostic stuff.
>
> Number of citations that contain the words "real time PCR" searched for on
> Google Scholar by Year
>
> 2012 47,400 (of these 23,900 include the word diagnostic)
> 2011 80,500
> 2010 104,000
> 2009 114,000 (of these 18,600 include the word diagnostic)
> 2008 113,000
> 2007 104,000
> 2006 85,100
>
>
> There is a reason the citations have dropped off drastically as a research
> tool because people have found it is not a really good technique.
>
>
>
> On Tue, May 21, 2013 at 3:52 PM, Josh Perfetto <jo...@snowrise.com> wrote:
>>
>> On Tue, May 21, 2013 at 8:58 AM, Jeswin <phill...@gmail.com> wrote:
>>>
>>> On Mon, May 20, 2013 at 7:05 PM, Josh Perfetto <jo...@openpcr.org> wrote:
>>> > Ashley, the plan is to provide a very low-cost unit, capable of single
>>> > channel detection in 16 200 uL PCR tubes. The machine will have fast
>>>
>>> Why not PCR plates like in reqular qPCR machine? Maybe your detection
>>> method differs so you can't use plates?
>>
>>
>> Hi Jeswin,
>>
>> Basically it was far cheaper to create this machine for 16 wells than for
>> 96 wells, and I wanted to introduce something at a very affordable price
>> range first. I think qPCR is a very powerful technique that too few people
>> do because the hardware is so expensive. For many applications you can not
>> only get quantitative data, but also avoid running gels which dramatically
>> increases your workflow.
>>
>> So at 16 wells it wouldn't be much of a plate, though of course the
>> spacing is the same so you could cut up your 96 well plates into 8 16 well
>> plates if you wanted. Or just use 8-tube strips.
>>
>> -Josh
>>
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--
-Nathan

[Josiah Zayner]

Are you talking about purifying the mRNA?

[Nathan McCorkle]

I'm talking about purifying the mRNA and further reactions.

> https://groups.google.com/d/msgid/diybio/b646acbc-85f4-48a4-8e01-a2f263a4e6c8%40googlegroups.com?hl=en.

[Josiah Zayner]

For purifying mRNA I have always used kits. I don't think it matters which one (I have used Stratagene, Qiagen, Promega with success). Then reverse transcribe with polydT primers. 

I think usually the biggest issue is with RNA purification and it is always good to run it out on a gel and see if you have a smear or you can see the ribosomal subunits and tRNA decently. 

[Nathan McCorkle]

Why not use normal specific primers rather than polydT (if you have
that much knowledge of the situation)? What about LN2 vs bead beating?

> https://groups.google.com/d/msgid/diybio/57abbd6e-1f6c-40e9-8b83-dda4aa0c398d%40googlegroups.com?hl=en.

[Josiah Zayner]

What are you trying to extract the mRNA from? 

If you are only looking for a specific gene then use specific primers but it seems more superfluous. 

If you are looking at mRNA and want to quantify you need a control, GAPDH or Actin or something.

So if you reverse transcribe with specific primers you need primers for your gene/genes of interest and controls and all. With a polyDT or random hexamer primer you turn everything into cDNA. 

I am talking about quantitating mRNA though. 

If you are just talking about looking for the presence of a gene then that is different. 

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[Nathan McCorkle]

I was working with animal tissue, lung tissue to be specific. I wasn't
interested in quantifying it or looking for the presence, I was simply
trying to get the transcript into DNA form and pull it out, to be used
in an expression cassette elsewhere.

> https://groups.google.com/d/msgid/diybio/CAEUkM4s_4feeXYh%2BPd2kOXE5bEGuygPjDCp5te9in0%3Dg-AF7PA%40mail.gmail.com?hl=en.