Second Round, Second strand cDNA synthesis ON ICE

11The 5X VILO™ Reaction mix already contains random primers, MgCl2, and dNTPs. The 10X SuperScript® Enzyme Mix includes the SuperScript® III Reverse Transcriptase (reduced RNase H activity and high thermal stability for extended synthesis), the RNAseOUT™ Recombinant Ribonuclease Inhibitor, and a helper protein proprietary to Invitrogen™.

Second strand synthesis if template is SS DNA.

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.

cDNA synthesis(From Nugen RNAseq V2) and Covaris Shearing

Also, thaw the Second Strand cDNA Synthesis reagents (yellow).

The PrimeScript First Strand cDNA Synthesis Kit contains all of the reagents necessary for synthesis of first-strand cDNA from total or poly(A)+ RNA using PrimeScript Reverse Transcriptase. Choose this cDNA synthesis kit when you want to synthesize full-length cDNA with provided primers (select from oligo dT or random 6-mer primers, both included in the kit.) This cDNA synthesis kit is powered by PrimeScript Reverse Transcriptase, which has exceptionally strong strand displacement activity and efficiently synthesizes cDNA. PrimeScript RTase is robust, versatile and well-suited for applications requiring full-length cDNA such as cDNA library construction. This PrimeScript-based cDNA synthesis kit also works well with other cDNA-dependent techniques including RT-PCR, cDNA probe preparation and real-time quantitative RT-PCR (qRT-PCR or RT-qPCR). The PrimeScript First Strand cDNA Synthesis Kit is ideal for reverse transcription of many different RNA templates, including GC-rich templates and RNAs with high levels of secondary structure.

[35] Second-strand cDNA synthesis: mRNA fragments …

The NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module has been optimized to generate double-stranded cDNA from first-strand cDNA, as part of the NEBNext Directional RNA library preparation workflow. This workflow uses the “dUTP” method for strand-specific library construction, which depends on incorporation of uracil into the second strand cDNA, enabled by the NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix, included in this module.

of the RNA during first-strand cDNA synthesis, ..

15The substitution of the dTTP by dUTP in the dNTP mix is critical in this protocol since it will allow using the USER™ (Uracil-Specific Excision Reagent) enzyme prior library amplification and achieving strand specificity. The USER™ enzyme will leave a nucleotide gap at the location of a uracil in the second strand of the cDNA.

Reaction Mix for Second Strand Synthesis a.

The first strand cDNA productgenerated is more than 10 kb (Figure 1).

General Information forSuccessful cDNA Synthesis:

Template RNA
Intact RNA of high purity is essential for sensitive RT-PCR detection.

NEBNext mRNA Second Strand Synthesis Module from …

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.