I am trying to use the Ovation RNA seq V2 kit from Nugen or Tecan to amplify low-input RNA (about 1ng). (https://lifesciences.tecan.com/ovation-low-input-rna-seq-kit-v2?p=tab--1).
The Protocol and workflow of the SPIA amplification process show linear amplification of cDNA, which is single-stranded (As per the schematic provided by them). However, the kit claims it generates double-stranded cDNA, which is not shown in the figure or explained how it is done!!!
Can somebody please help me understand this?
How does the Ovation RNA seq v2 kit generate double-stranded cDNA instead of single-stranded cDNA??!!
Nugen/ tecan suggests using their library preparation kits (for Illumina) which can utilize only dsDNA templates but not single-stranded.
They seem to work well, but I don't understand how the dsDNA is generated in the amplification step (RNA Seq V2 kit).
Please help.
On Page 1 of the introduction of the SPA protocol (https://labettor.com/uploads/products/protocols/1028.pdf):
Fragmentation of the mRNA within the cDNA/mRNA complex creates priming sites for DNA polymerase to synthesize a second strand, which includes DNA complementary to the 5’ unique sequence of the first strand chimeric primers. The result is a double-stranded cDNA with a unique DNA/RNA heteroduplex at one end.
Thank you, Wilhelm,
That still did not address the main question, though. Please check again.
After 2nd strand synthesis step, there is an SPIA amplification step, SPIA primer can bind to only the second strand and amplify only that strand. The first strand does not have the SPIA primer binding site, so it does not get amplified.
This may lead to a high quantity of single-stranded cDNA (due to SPIA amplification) and very low double-stranded cDNA if at all there is any.
So the question is, How did the manufacturer claim that the product will be double-stranded cDNA? whereas whatever is shown in the diagram, can generate only one strand.
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