I know that there are two disadvantages of using sequence specific primers. I do not quite understand the disadvantages.
First one is "only one assay can be run from each cDNA reaction." I am not sure why this is the disadvantage. And the other one is, only one real-time assay can be run. Again, I do not know why this poses as the disadvantage.
I have an introductory understanding of realtime PCR.
Apparently your question most likely concerns cDNA synthesis (reverse transcription) reactions and which priming strategy to use. This is where the problem that Eric above cites could arise if you subsequently perform qPCR with cDNA made with only a pair of sequence-specific primers.
When using only a sequence-specific primer pair (a forward and a reverse primer) during cDNA synthesis (from total RNA or mRNA) prior to qPCR, you only create a first strand cDNA of the target defined by the reverse primer run-off product (the forward primer does not participate here), and thus have limited yourself to only pursuing the amplification of that one particular target transcript during subsequent qPCR.
However, if you prime your RT reactions with a mixture of oligo [dT]n and randomers (e.g., hexamers, nonamers, pentadecamers etc.), then the entire transcriptome is converted to cDNA and all targets are present in the form of first-strand cDNA prior to the qPCR. Using only oligo [dT]n or anchored-dT primers will often give you "3' bias" which must be addressed by designing your qPCR primers toward the 3' region of the target sequence. But, when you use an appropriate mixture of oligo [dT]n and randomers, you cover the 5' ends of all transcripts as well - and are thus freer to design primers anywhere along the length of the mRNA [cDNA] transcripts for qPCR purposes.
All of the above applies only to "two-step qPCR" - wherein cDNA is synthesized first, and then, after that, in a second step: the qPCR is performed on the prepared cDNA at optimal dilutions of that cDNA.
On the other hand, with "one-step qPCR", one merely adds total RNA or mRNA (at an optimal, already empirically known/discovered non-inhibitory dilution) to the qPCReaction wherein the mastermix contains both an RT enzyme (RNA-dependent DNA polymerase), and a DNA-dependent DNA-polymerase enzyme (e.g., Taq), and sequence-specific primers only. It is here, in the one-step qPCR application, where there is an absolute requirement of using sequence-specific primers only with no need for other priming whatsoever. Given ample total RNA of high quality, one can run many targets at once. I frequently run up to 15 or 20 different targets at once on multiple samples (>25) using this one-step approach. I run my one-step qPCR immediately after RNA isolation, DNAse treatment, and proper RNA dilution to avoid any degradation of RNA due to storage. And the RNA I use in this application always demonstrates RIN of >=8.5 on an Agilent Bioanalyzer 2100. The RNA I prepare and use is still useful 3+ years after isolation due to the way I prepare and store it (proper use of nuclease-free water and the very expensive RNAseOUT reagent is key here).
The big caveat in the two-step approach has always been in the possibility that the snapshot of the transcriptome may be skewed depending on the priming strategy and the choice of reverse transcriptase enzyme used for RT. And, according to stalwart publications by Bustin and Nolan et al., certain mRNA transcripts are more able to be reverse-transcribed than others during RT...which can really warp the playing field so-to-speak. In recent world-wide meetings on qPCR methodologies, it is widely felt that the least egregious approach to RT priming (in two-step qPCR) is using an appropriate mixture of olgo [dT]n and randomers.
cDNA does store better than RNA in most situations, and therein lies the strength in the two-step approach - even though the snapshot may be warped to various degrees in ways yet unknown...
The disadvantages are costs and comparison of gene expression levels for multiple genes. When you use oligo-dT or anchored-dT primers you can use the cDNA for several (real time) assays. Since the input (cDNA) will be the same for these reactions, this allows a better comparison of expression levels for several target genes, ie comparison to reference genes for normalisation.
Apparently your question most likely concerns cDNA synthesis (reverse transcription) reactions and which priming strategy to use. This is where the problem that Eric above cites could arise if you subsequently perform qPCR with cDNA made with only a pair of sequence-specific primers.
When using only a sequence-specific primer pair (a forward and a reverse primer) during cDNA synthesis (from total RNA or mRNA) prior to qPCR, you only create a first strand cDNA of the target defined by the reverse primer run-off product (the forward primer does not participate here), and thus have limited yourself to only pursuing the amplification of that one particular target transcript during subsequent qPCR.
However, if you prime your RT reactions with a mixture of oligo [dT]n and randomers (e.g., hexamers, nonamers, pentadecamers etc.), then the entire transcriptome is converted to cDNA and all targets are present in the form of first-strand cDNA prior to the qPCR. Using only oligo [dT]n or anchored-dT primers will often give you "3' bias" which must be addressed by designing your qPCR primers toward the 3' region of the target sequence. But, when you use an appropriate mixture of oligo [dT]n and randomers, you cover the 5' ends of all transcripts as well - and are thus freer to design primers anywhere along the length of the mRNA [cDNA] transcripts for qPCR purposes.
All of the above applies only to "two-step qPCR" - wherein cDNA is synthesized first, and then, after that, in a second step: the qPCR is performed on the prepared cDNA at optimal dilutions of that cDNA.
On the other hand, with "one-step qPCR", one merely adds total RNA or mRNA (at an optimal, already empirically known/discovered non-inhibitory dilution) to the qPCReaction wherein the mastermix contains both an RT enzyme (RNA-dependent DNA polymerase), and a DNA-dependent DNA-polymerase enzyme (e.g., Taq), and sequence-specific primers only. It is here, in the one-step qPCR application, where there is an absolute requirement of using sequence-specific primers only with no need for other priming whatsoever. Given ample total RNA of high quality, one can run many targets at once. I frequently run up to 15 or 20 different targets at once on multiple samples (>25) using this one-step approach. I run my one-step qPCR immediately after RNA isolation, DNAse treatment, and proper RNA dilution to avoid any degradation of RNA due to storage. And the RNA I use in this application always demonstrates RIN of >=8.5 on an Agilent Bioanalyzer 2100. The RNA I prepare and use is still useful 3+ years after isolation due to the way I prepare and store it (proper use of nuclease-free water and the very expensive RNAseOUT reagent is key here).
The big caveat in the two-step approach has always been in the possibility that the snapshot of the transcriptome may be skewed depending on the priming strategy and the choice of reverse transcriptase enzyme used for RT. And, according to stalwart publications by Bustin and Nolan et al., certain mRNA transcripts are more able to be reverse-transcribed than others during RT...which can really warp the playing field so-to-speak. In recent world-wide meetings on qPCR methodologies, it is widely felt that the least egregious approach to RT priming (in two-step qPCR) is using an appropriate mixture of olgo [dT]n and randomers.
cDNA does store better than RNA in most situations, and therein lies the strength in the two-step approach - even though the snapshot may be warped to various degrees in ways yet unknown...
Thank you for the appropriately detailed answer.
Could you just tell me what a "reverse primer run-off product" is??
When performing one-step qPCR, during the RT phase, the sequence-specific reverse primer primes the mRNA to make cDNA during the RT reaction since the mRNA is 'sense' in orientation (by definition; it is a 'carbon copy' of the DNA 'sense' coding strand; although mRNA has U's instead of T's of course), only the reverse primer can complement the mRNA sequence since the reverse primer is 'anti-sense' (thus the name 'reverse'). The reverse transcriptase (RT) enzyme goes as far as it can to make cDNA from the 3'-end of the reverse primer before it falls off either due to fidelity of the enzyme itself, or secondary structure in the RNA template.
Normally, we expect good RT enzymes to go at least 1200 nt before it 'aborts' mission, and 1200 is usually sufficient for qPCR as qPCR likes 80 to 150 bp amplicons for the most part (although up to 400 bp amplicons work fine with SYBR Green-based qPCR in many cases). Anyway, the 'run-off' product I was referring to is the first-strand (single stranded) cDNA that the RT enzyme is able to transcribe into cDNA using the sequence-specific reverse primer annealed to the RNA template to initiate the reverse transcription.
This first-strand cDNA can be of a number of lengths (again, depending on the factors stated above). But, after the 5th cycle in the PCR phase of the one-step qPCR, the desired blunt-end transcripts defined by both forward and reverse primers predominate as the double-stranded amplicon product in the reaction (accruing geometrically/exponentially: 1, 2, 4, 8, 16, 32, 64, 128...), while the first-strand cDNA transcripts quickly become a miniscule portion of all the single transcripts being made (since the first-strand 'run offs' only accrue arithmetically in the reaction: 1,2,3,4,5,6,7... or 2, 4, 6, 8,10,12 etc., since only the forward primer is being used on the run-off PCR (the run-off cDNA: being 'anti-sense' or 'complementary' to the original 'sense' mRNA, can only anneal to the sequence-specific forward primer). I am using a typical eukaryotic situation to state these things here - to keep it simple. I hope that helps. If you think about it a bit, it makes 'sense.'
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