Ovation RNA seq v2 kit, SPIA amplification

[praveenhajeri]

How does the Ovation RNA seq v2 kit generate double-stranded cDNA instead of single-stranded cDNA??!!


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.

[Ben3]

Hello praveenhajeri, there is a second PCR step (Second Strand cDNA Synthesis) in the protocol that provides a complementary strand of the cDNA making it double-stranded.

It's listed in the thermocycler program in the protocol (https://www.tecan.com/hubfs/HubDB/Te..._System_V2.pdf).

[praveenhajeri]

Hi Ben3 ,
Thanks,

But the question is much deeper than what you mentioned. Please have a look at it again and help me out.

As per the figure, a large amount of double-stranded cDNA is synthesized in the SPIA step but not in the 2nd strand synthesis step.

Manufacturers say that it generates double-stranded cDNA, but the SPIA primers are shown to bind to only 2nd-strand cDNA and produce large-scale products (as per the figure it is only one strand). That means the product of SPIA is only one strand and is complementary to 2nd strand cDNA (because only that has SPIA primer binding sequence).

So the question is, how does it produce a large quantity of double-stranded cDNA?

First-strand cDNA and second-strand cDNA will be in very low quantity, and they can't make the bulk of ds cDNA. That is why SPIA amplification is needed. Also, Both won't be amplified in the SPIA step (as per the figure) because only one of them has SPIA primer binding sites.

So the question is, how does it produce double-stranded cDNA?

[Ben3]

Hello praveenhajeri

You're correct it doesn't explain it very well. But they do a slightly more detailed explanation of SPIA in this paper:

https://academic.oup.com/clinchem/article/51/10/1973/5629509?login=false#F1

Skip to the Results and Discussion section and they explain the amplification process in the first few paragraphs of that section.

[praveenhajeri]

Hi Ben3,
Thanks,

I have seen that paper!
In fact, the figure I posted in my original question is the same as in that paper. (just that the figure I posted is in color.)

That paper clearly mentions...SPIA produces single stranded cDNA but not dsDNA.
the exact quote from the paper is "The amplification products are single-stranded cDNA suitable for a variety of detection and quantification platforms, "

Ovation RNA Seq V2 kit uses this method but claims to generate double-stranded cDNA. But it does not explain how?

My question is to understand why and how is it done!?