Re: [DIYbio] Re: Reverse Transcriptase PCR

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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>> >> >
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>> >>
>> >> --
>> >> -Nathan
>> >
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>>
>> --
>> -Nathan
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--
-Nathan

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