iScript Explore One-Step RT and PreAmp Kit

Preamplification does not introduce bias. Potential for bias was evaluated using External RNA Consortium Controls (ERCC) controls, which are RNA transcripts of a known copy number. ERCC concentrations from single copy to 16,000 copies were used to assess bias across a wide range of transcript concentrations. RNA samples were reverse transcribed using iScript™ Advanced cDNA Synthesis Kit or reverse transcribed and preamplified using iScript Explore One-Step RT and PreAmp Kit. The R² value of 0.952 indicates a high correlation between samples with and without preamplification, demonstrating that preamplification does not introduce bias.

1 copy (average) input
iScript Explore: 20 of 20 positive
iScript Advanced: 2 of 20 positive
Competitor T: 1 of 20 positive

iScript™ Explore One-Step RT and PreAmp Kits enable sensitive detection of rare transcripts. The unique single-step RT and preamplification reaction allows for reproducible detection of targets down to the single-copy level.

RNA samples containing a single copy of target RNA were reverse transcribed using iScript Advanced cDNA Synthesis Kit or Competitor T Reverse Transcriptase, or reverse transcribed and preamplified using iScript Explore One-Step RT and PreAmp Kit. Resulting cDNA samples were run in 20 replicate qPCR reactions to determine target detection. Only the iScript Explore One-Step RT and PreAmp Kit provides consistent detection of the rare target in each qPCR reaction (20 of 20 reactions were positive). Rare events were not reliably detected in the absence of preamplification (resulting samples from iScript Advanced cDNA Synthesis Kit and Competitor T Reverse Transcriptase). Melt curve analysis demonstrates a single peak in all positive samples, suggesting that a single, specific product was produced in all cases.

The PreAmp Control Assay validates the performance of the preamplification reaction when using the iScript™ Explore One-Step RT and PreAmp Kit. This control provides confidence that preamplification was completed as expected.

This control is included on all PrimePCR lncRNA Predesigned Arrays and can also be included in any custom designed PrimePCR Array. When using iScript Explore Kit, PAQ1, but not PAQ2, is amplified during the preamplification reaction. During the qPCR reaction, both PAQ1 and PAQ2 are amplified. The delta Cq between the preamplified and the non-preamplified assays should approximately equal the number of preamplification cycles run.

PrimePCR PreAmp Control demonstrates preamplification efficacy: Fourteen rounds of cDNA synthesis and preamplification were performed using the iScript Explore One-Step RT and PreAmp Kit followed by qPCR. Preamplification efficacy was determined by analyzing the delta Cq between the PAQ1 and PAQ2 assays. The delta Cq between these two reactions was equivalent to the number of preamplification cycles performed.

iScript™ Explore One-Step RT and PreAmp Kit effectively clears contaminating genomic DNA (gDNA). HeLa RNA was purified using the Aurum Total RNA Mini Kit without gDNA clearance. RNA (10 ng) was processed using iScript Explore One-Step RT and PreAmp Kit with (red traces) and without (green traces) the gDNA clearance option for 14 preamplification cycles. Levels of residual gDNA were assessed using the PrimePCR™ gDNA Contamination Control Assay. Samples treated with DNase showed no detectable expression, illustrating complete gDNA clearance.

Sso7d, a 7 kD protein obtained from Sulfolobus solfataricus, binds to dsDNA without any sequence specificity.

Sso7d is covalently linked to a DNA polymerase and is able to stabilize the polymerase-template complex without compromising the structural integrity, thermal stability, or catalytic activity of the enzyme.

The unique structure of this Sso7d fusion polymerase provides several significant advantages over ordinary DNA polymerase:

• Increased processivity
• Effective amplification in GC-rich regions or strong secondary structure
• Efficient amplification of longer products
• Superior tolerance to PCR inhibitors