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Archived Comments for: Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos

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  1. Two important steps to improve the sensitivity, precision and accuracy of SUPeR-seq method

    P.A. Desingu, Indian Veterinary Research Institute

    30 July 2015

    SUPeR-seq method is published in this journal, is need some suggestion to improve the technique. Here, I would like to suggest two events.

     

    First, Poly (A) tails addition to first strand cDNA step utilized simultaneously dATP and ddATP concentrations of 3 mM and 30 μM, respectively. The ddATP concentration is less, even though it has possibility to terminate early Poly (A) tails addition. The early termination of Poly (A) tails makes the first strand cDNA unavailable for second strand cDNA synthesis. This method is for single cell cDNA amplification; very less amount expressed mRNA may missed by this method. I would like to suggest that split the step into two; one is with dATP and next with ddATP.

     

    Second, the PCR reaction was carried out with NH2-AnchorX-T15 and NH2-AnchorY-T24 primers. Both of the primers have Poly (T) at 3’ end; they possibly compete for both strands (first and second strand) cDNA in PCR reaction. Even though annealing temperature is high, totally this would not avoidable. This reaction leads to getting false antisense/sense RNA sequences in sequencing results. Here, I would like to suggest that using AnchorX and AnchorY with out Poly (T) will avoid the false antisense/sense RNA sequences in sequencing results.

     

    This modification is expected to improve the sensitivity, precision and accuracy of SUPeR-seq method.

     

     

    Competing interests

    Competing interests

     I have no competing interest

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