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 8:58 AM, Jeswin <phillyj101@gmail.com> wrote:
On Mon, May 20, 2013 at 7:05 PM, Josh Perfetto <josh@openpcr.org> wrote:Why not PCR plates like in reqular qPCR machine? Maybe your detection
> 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
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--To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CA%2BL%3DET2ohCCVMULKPdouGQ5KSuB1TOYr0gB8G%3DZUriYcPtMejg%40mail.gmail.com?hl=en.
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