To ensure that amplifiable material was present in all specimens and to avoid false-negative results, real-time amplification of the reference gene PBGD was performed for all samples. To reduce the risk of contamination, each procedure such as RNA extraction, cDNA synthesis, preparation of the RT-qPCR reactions and thermocycling, were performed in separate rooms while preparation of the cDNA and PCR reactions were set up in different PCR-hoods. A positive (cell line cDNA) and a negative control (H2O) were included in all runs.
Our preliminary results on the comparison of RT-qPCR with the CellSearch in the adjuvant setting for a limited number of patients, have shown a higher positivity rate in favor of RT-qPCR, despite the fact that we have used the same amount of peripheral blood and positive immunomagnetic selection through EpCAM. This could be possibly explained by the fact that we are additionally isolating mRNA through oligo-dT beads and thus we are using the whole isolated mRNA fraction for cDNA synthesis, in combination with the superior sensitivity of RT-qPCR, as verified at the 10 copies/μL level. To elucidate this, we plan to compare RT-qPCR and CellSearch in the adjuvant setting for a large number of patients in the near future. Consistent with our findings, in a recent study it was reported that there was no association in the detection rates of CTCs from patients with breast cancer when the same samples were analyzed by both the CellSearch and the AdnaTest: 18.4% of patients with primary breast cancer were positive by CellSearch and 35.7% by the AdnaTest .
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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.
The first strand cDNA productgenerated is more than 10 kb (Figure 1).
General Information forSuccessful cDNA Synthesis:
Intact RNA of high purity is essential for sensitive RT-PCR detection.
In general 1 ng to 1 μg total RNA or 0.1-100 ng mRNA arerecommended.
First Strand cDNA SynthesisReaction
Denaturation of RNA and primer at 70°C for 5 minutes canremove secondary structures that may impede long cDNA synthesis.
The human mammary carcinoma cell lines SKBR-3, MDA-MB-231 and MCF-7 were used for the development of the assay and the generation of gene specific quantification calibrators. Cells were counted in a hemocytometer and their viability was assessed by trypan blue dye exclusion. Serial dilutions of a known number of cells corresponding to 1-1000 cells, for which total RNA isolation and cDNA synthesis was performed, were prepared. These cDNAs were kept in aliquots at -20°C and used for the validation of the assay, prior to the analysis of patient's samples.
Total RNA-Seq differs from other transcriptome sequencing strategies as the method allows for sequencing of both coding and non-coding RNA. Rather than undergoing polyA+ selection that targets the mRNA species, the whole RNA is often subjected to rRNA depletion instead. As rRNA makes up a large percentage of the entire RNA, reduction of rRNA helps allocate more sequencing reads to transcripts of interest. cDNA is synthesised from the rest of the rRNA depleted RNA. Optionally, strand-specific information during downstream processing can be provided. The cDNA is then treated as in all other RNA-Seq workflows.
For example, 5minutes incubation is enough for a 2 kb cDNA synthesis.
Choice ofPrimers for Reverse Transcription
Oligo d(T) priming is preferredfor most applications because it ensures that all cDNA copies terminate at the3´ end of the mRNA and produces the longest contiguous cDNA.
1. It depends on how much starting material you have and the primers/reverse transcriptase that you use. But basically smears are normal. Even when the rRNA is not visible.
2. I have never compared both, but I presume both would also be smears on the gel, as both would still produce varying lengths of cDNA.
3. The smear is fine. Just use the cDNA in a PCR to check, or run a spectro.
4. I would say it depends how many templates the primers stick to. The primer annealing step of 70C for 5 min prior to addition of reverse transcriptase decides how many templates have primers. Since there are no cyclic temperatures, the MMLV can only continue producing cDNAs from mRNAs (at 37C or 42C) that have primers annealed. So basically it's one RNA one cDNA.
Random Primer Mixoffers good performance in a wide range of RNA templates.
When agene-specific primer is used in a cDNA synthesis reaction, the cDNA product canbe used only for amplification of that transcript.