Re: [DIYbio] Re: Reverse Transcriptase PCR

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.

On Sun, May 26, 2013 at 1:14 PM, Josiah Zayner <josiah.zayner@gmail.com> wrote:
> 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.
>
>
> On Sun, May 26, 2013 at 2:55 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
>>
>> Why not use normal specific primers rather than polydT (if you have
>> that much knowledge of the situation)? What about LN2 vs bead beating?
>>
>> On Sun, May 26, 2013 at 11:37 AM, Josiah Zayner <josiah.zayner@gmail.com>
>> wrote:
>> > 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.
>> >
>> >
>> > On Friday, May 24, 2013 4:47:31 AM UTC-5, Nathan McCorkle wrote:
>> >>
>> >> I'm talking about purifying the mRNA and further reactions.
>> >>
>> >> On Thu, May 23, 2013 at 8:18 AM, Josiah Zayner <josiah...@gmail.com>
>> >> wrote:
>> >> > Are you talking about purifying the mRNA?
>> >> >
>> >> >
>> >> > On Tuesday, May 21, 2013 5:30:08 PM UTC-5, Nathan McCorkle wrote:
>> >> >>
>> >> >> 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
>> >> >
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-Nathan

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