Description

This product is a 2 × Master Mix for "1-step real-time PCR" using a thermostable DNA polymerase derived from Thermus thermophilus (Tth) HB8 (1). Tth DNA polymerase exhibits reverse transcriptase activity in the presence of Mn2+ ions. This system allows for "1-step real-time PCR", including reverse transcription and PCR steps. RNA-direct™ Realtime PCR Master Mix is applicable for TaqMan® assay or hybridization probe assay, in combination with each probe. RNA-direct™ SYBR Green® Realtime PCR Master Mixcan be applied to an intercalation assay with SYBR® Green I.

Features

  • Suitable for high-throughput real-time PCR and increases reliability of product, due to lowered risk of contamination.
  • Can be used in systems using glass capillaries (e.g., LightCycler, Roche Molecular Systems, Inc.).
  • Can be used in systems using passive reference (e.g., ABI PRISM® 7700, Applied Biosystems, Inc.).
  • Hot Start technology, using anti-Tth DNA polymerase antibodies, allows for high specificity and reproducible amplification.

Applications

<RNA-direct Realtime PCR Master Mix>
TaqMan® assays or hybridization probe assays

<RNA-direct SYBR® Green Realtime PCR Master Mix>
Intercalation assay with SYBR® Green I

Storage condition

Store at -20ºC

Components

This reagent includes the following components for 100 reactions, with a total of 50 µL per reaction. All reagents should be stored at -20ºC.

<QRT-101>

RNA-direct™ Real-time PCR Master Mix 0.5 ml x 5
50 mM Mg(OAc)2 0.5 ml x 1

<QRT-101>

RNA-direct™ SYBR® Green Realtime PCR Master Mix 0.5ml x 5
50mM Mg(OAc)2 0.5ml x 1

Application data

Example 1.Correlation between 1-step and 2-step methods

Correlation between the 1-step method (RNA-direct™ SYBR® Green Realtime PCR Master Mix [Code No. QRT-201]) and conventional 2-step method (ReverTra Ace® [Code No. TRT-101] (highly efficient reverse transcriptase)->SYBR Green® Realtime PCR Master Mix [Code No.QPK-201]) was evaluated based on the expression of Protein kinase δ (PKCδ) mRNA. The results from the 1-step method were found to be highly correlated with those of the 2-step method (Fig. 1).

Fig. 1 Comparison of the quantitation of target mRNA

The Y-axis indicates the relative quantitation value of Protein kinase δ (PKCδ) mRNA
Yellow bar (left): 1-step method using 50 ng total RNA
Yellow bar (right): 1-step method using 200 ng total RNA
Blue bar: 2-step method

Example 2.Comparison of sensitivity

Amplification of G3PDH mRNA was detected using serially diluted poly(A)+ RNA (10n dilution) with SYBR® Green Realtime PCR kits including Tth DNA polymerase. The RNA-direct™ SYBR® Green Realtime PCR Master Mix [Code No. QRT-201)] showed greater sensitivity and signal intensity than the other kit (company A).

Fig. 2 Comparison of the SYBR® Green assay

References

  • everse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Myers T. W. and Gelfand D. H. , Biochemistry, 30: 7661-6 (1991)