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First strand cDNA synthesis kit

[NIPPON GENE] GeneAce cDNA Synthesis Kit

GeneAce cDNA Synthesis Kit is a kit for synthesizing first strand cDNA using RNA as a template.
Because it uses "GeneAce Reverse Transcriptase," an M-MLV-derived (RNase H-) reverse transcriptase, long-chain cDNA can be synthesized efficiently. In addition, the template RNA, which may inhibit the PCR, is degraded by RNase H treatment following the reverse transcription reaction.

Features

  • Contains all reagents required for cDNA synthesis
  • More efficient, full-length cDNA synthesis
  • More efficient RT-PCR thanks to RNase H treatment

Principles

Synthesis of first strand cDNA and removal of RNA strand

When the amplification efficiency of RT-PCR is poor, RNase H treatment may result in greater PCR efficiency.

Example of an experiment

Comparison of efficiency of long-chain cDNA (10 kb) synthesis

cDNA was synthesized using an oligo (dT) primer with total RNA (2 µg) from mouse FM3A cells as a template. Real-time PCR was performed using the obtained cDNA as a template, and the quantity of utrophin gene cDNA was compared. (the primer was designed approximately 10 kb from the 3' end)

Real-time PCR reagent:GeneAce SYBR® qPCR Mix α Low ROX
Target:Utrophin gene (approximately 10 kbp)

Results: This product was shown to synthesize long-chain cDNA more efficiently than the competitor's products.

Effect verification of RNase H treatment

cDNA was synthesized using this product with total RNA (2 µg) from HeLa cells as a template. Using the obtained cDNA as a template, PCR efficiency was compared with and without RNase H treatment.

  • With RNase H treatment (Lane +): PCR products when RNase H treated cDNA (single-stranded DNA; 37℃ for 15 min) was used as a template
  • Without RNase H treatment (Lane -):PCR products when the obtained cDNA (heterozygous double-stranded DNA-RNA) was used directly as a template
  • Marker (Lane M): Gene Ladder Wide 1 (Product Number: 313-06961)

Target:Part of the EPAS1 gene (approximately 2.6 kb)

Results: RNase H treatment resulted in greater PCR efficiency.

cDNA synthesis from total RNA and real-time PCR

cDNA was synthesized using this kit with total RNA from HeLa cells as a template. Real-time PCR was performed by serially diluting the obtained cDNA.

RNA extraction kit:ISOSPIN Cell & Tissue RNA (Product Number: 314-08211)
Real-time PCR Reagen:GeneAce Probe qPCR Mix Ⅱ (Product Number: 313-08823)
Template:Serially diluted cDNA (RNA equivalent: 10 ng, 1 ng.100 pg, 10 pg, 1 pg)
Target:Part of the β-actin gene
PCR conditions:(System: ABI7500) 95 ℃ for 10 min → [95 ℃ for 30 sec → 60 ℃ for 1 min]×45 cycles

Results: cDNA synthesis from total RNA and real-time PCR were successfully performed

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Reverse Transcriptases

Real-time PCR Reagents

RNA Extraction/Purification Reagents

Product content may differ from the actual image due to minor specification changes etc.

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