Dear Researcher
I am trying to quantify a gene in 2 sample sets one of which yields a consistent discernible Ct value, albeit at the low end of meaningful detection (>35cycles) and one of which is even lower in expression and consequently fails to consistently yield a detectable Ct value (within 40 cycles)
We are hoping to compute relative expression by delta delta Ct but to do that we need consistent Ct values for both sample sets below 40 cycles
Thus far we have performed assays using Taq Man probes where there is limited scope for optimisation
Consequently, were are about to evaluate sybr green qPCR where there is more scope to do things like primer titrations (which I have done successfully in the past)
Allied to the above I was thinking about and would invite comments on the following as well (which I havn't really evaluated):
1. Use of Additives like betaine in the cDNA synthesis (RT) step (even though the issue is not secondary structure in the mRNA target limiting primer efficiency from what we can ascertain) and use of different polymerases like ss IV ?
2. cDNA preamplification: I have read about about users performing (in effect) PCR on cDNA samples prior to actual qPCR with TaqMan probes and/or sybr green or the use of pre amplification mixes by companies like Roche, Abi and Qiagen prior to qPCR. Can this be done to simply increase qPCR sensitivity without bias (rather than simply boost cDNA from compromised i.e. degraded and/r FFPE material)
Can anybody comment on the effectiveness of such strategies for increasing qPCR sensitivity as opposed to standard ways like primer titration in the qPCR reactions themselves; Which obviously are preferable and I will try as well
https://www.researchgate.net/post/Does_anyone_have_experience_on_cDNA_preamplification_systems_for_RT_qPCR_application
Hi Laurence,
Adding MS-2 carrier RNA ('cRNA') to your sample diluent can stave off target DNA binding to plastic/qPCR plate wells during cycling. One would add a stray carrier nucleic acid like this since target DNA/cDNA template may apparently be unable to participate in the PCR due to unwanted electrostatic interaction of its negatively-charged phosphate backbone with positively-charged plastic surfaces. This unwanted effect of course becomes even more pronounced with low copy template reactions (such as the ones you are dealing with here). Colleagues of mine have noted dramatic improvement in their qPCR sensitivity doing this (adding cRNA to the sample diluent). Also, (if possible) double the reaction sizes so you can at least add twice the amount of sample per reaction (although the cost goes up then). This would help you improve by about 1 Cq value - but perhaps that is still helpful in the 35 to 40 Cq range of the assay. I too am ambivalent about pre-amplification strategies, as one never knows the bias it introduces.
Kappa Biosystems sells a very aggressive Taq polymerase that may also wake things up for you - although I am not an advertiser for companies, I have seen this particular polymerase beat many competitors by about 3 Cq on the same sample sets. But changing reagents mid-stream is always a hard-sell.
Also, apparently adding a tone (~100 Hz I believe the frequency was) during RT reactions apparently improves cDNA yield). The tone is about two G#'s below middle C on the piano.
https://www.youtube.com/watch?v=mejO0hV_w80
(although 2 G#'s below middle C is actually ~103.826 Hz).
Acoustic microstreaming:
http://www.biotechniques.com/BiotechniquesJournal/2011/February/Acoustic-microstreaming-increases-the-efficiency-of-reverse-transcription----reactions-comprising-single-cell-quantities-of-RNA/biotechniques-308394.html
And:
http://www.biotechniques.com/BiotechniquesJournal/2011/February/BioSpotlight/biotechniques-43791.html
Basically this is co-opting the growing field of cymatics to accomplish physical ends.
Perhaps just sound itself would help - without the need for cRNA... don't know yet.
I double-checked one of the publications listed above - and it is a 150 Hz tone they used for micro-mixing by sound.
This frequency is between the first D and a D# (below middle C).
D of 146.832 Hz and a D# of 155.563 Hz at this spot on the piano.
A link to the tone at 150 Hz:
https://www.youtube.com/watch?v=OVu99mgBtok
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