Re: [DIYbio] Re: Reverse Transcriptase PCR

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