Total RNA, including miRNAs, was polyadenylated andreverse-transcribed with a poly(T) adapter into cDNAs for real-time PCRusing the miRNA-specific forward primer and the sequence complementaryto the poly(T) adapter as the reverse primer.
SMART (Switching Mechanism at 5’ End of RNA Template) is a unique technology that allows the efficient incorporation of known sequences at both ends of cDNA during first strand synthesis, without adaptor ligation. The presence of these known sequences is crucial for a number of downstream applications including amplification, RACE, and library construction. While a wide variety of technologies can be employed to take advantage of these known sequences, the simplicity and efficiency of the single-step SMART process permits unparalleled sensitivity and ensures that full-length cDNA is generated and amplified.
Our portfolio covers a broad range of applications including, real-time PCR, endpoint PCR, high fidelity PCR, hot start PCR, long PCR, PCR direct from crude samples and molecular diagnostic PCR.
These findings constitute the first reportdescribingthe rigorous normalization of miRNA qRT-PCR data and have importantimplicationsfor proper experimental design and accurate data interpretation.
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These miRNAs were statisticallysuperior to the most commonly used reference RNAs used in miRNAqRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA.
miRNA microarray data from dozens of normal anddisease human tissues revealed ubiquitous and stably expressednormalizationcandidates for evaluation by qRT-PCR.
This study describes the identification andcharacterization of appropriate reference RNA targets for thenormalization of microRNA (miRNA) quantitative RT-PCRdata.
We then validated the results for 89 miRNAs byreal-time RT-PCR and challenged the use of this assay as a "goldstandard." Finally, we implemented a novel method to evaluatefalse-positive and false-negative rates for all methods in the absenceof a reference method.
When synthesizing a protein, DNA is transcribed into mRNA which is then translated into a protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes often contain introns which are not coding sequences, in contrast with exons, which are DNA coding sequences. During transcription, intronic RNA is excised from the RNA primary transcript and the remaining pieces of the RNA primary transcript are spliced back together resulting in processed mRNA. The mRNA code is then translated into an amino acid chain that comprises the newly made protein.
Using reverse transcriptase polymerases, DNA can be synthesised from mRNA and total RNA. Thus it is a 'complementary' copy of the mRNA, and is thus called complementary DNA (cDNA). cDNA forms the substrate for the majority of qPCR gene expression experiments.
In contrast to presenting a stepwisedescription of different platforms, we discuss expression profiling ofmature miRNAs by qPCR in four sequential sections: (1) cDNA synthesis;(2) primer design; (3) detection of amplified products; and (4) datanormalization.
Within this exciting field of research,whole-genome RT-qPCR based miRNA profiling in combination with a globalmean normalization strategy has proven to be the most sensitive andaccurate approach for high-throughput miRNA profiling (Mestdagh et al.,Genome Biology, 2009).
To correct for systematic variables such as amount of startingtemplate, RNA quality and enzymatic efficiencies,RQ-PCR data is commonly normalised to an endogenouscontrol (EC) gene, which ideally, is stably-expressed across the testsampleset.
Can you provide primers for sequencing/standard PCR/microarray?
No, unfortunately not. We concentrate on the one thing that we are best in the World at: Real-time PCR.