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There has been two separate steps in general RT-PCR process. cDNA are first synthesized with using reverse transcriptase and then implements PCR with
using DNA polymerase in the separate tube. However this method has a couple of drawbacks. It is quite inconvenient and easy to be have a cross contamination
by carry over of sample.To relieve these problems, iNtRON Biotechnology designed ONE-STEP RT-PCR PreMix Kit which can implement first-strand cDNA synthesis
and PCR reaction from RNA or mRNA template in one tube containing both reverse transcriptase and DNA polymerase.
ONE-STEP RT-PCR PreMix kit is more convenient than the conventional kit and more sensitive and specific amplification products can be taken.
Reverse transcriptase, as a DNA polymerase depending on RNA, generates a complementary DNA (cDNA) to RNA. Reverse transcriptase has a ribonuclease H activity.
Reverse transcriptase decomposes only RNA from RNA-cDNA hybrid which is made after finishing the reverse transcription reaction and only cDNA is left in the end.
Such a reverse transcriptase is extracted from retroviruses such as avianmyeloblastosis virus(AMV), Moloney murine leukemia virus(MMLV) and human immuno-deficiencyvirus(HIV).
Those reverse transcriptase are called as AMV RT, MMLV RT and HIV-RT and known to have a low cDNA synthesis efficiency due to low thermostability.
iNtRON’s ONE-STEP RT-PCR PreMix Kit is using OptiScriptTM RT System. It is optimal to amplify the template RNA which has a secondary structure RNA and performs
the best amplification regardless of the amount of template RNA.
ONE-STEP RT-PCR PreMix Kit is designed to implement first-strand cDNA synthesis and PCR reaction in one tube. It is selecting the OptiScriptTMRT System
and is suitable for expression of the low copy gene because OptiScriptTM RT System can guarantee the precise and high efficiency of reverse transcription
reaction from 1pg to 2mg template RNA. It is also using i-StarTaq™ DNA polymerase so that PCR reaction from non-specific primer or primer-dimer is blocked by hot-start PCR.
This kit contains a special stabilization material and the activities of enzyme (OptiScriptTM RT System, i-StarTaqTM DNA polymerase) last long enough.
It has also very convenient and simple protocols so that it implements the best cDNA synthesis and PCR reaction.
Even beginners can easily get high quality amplification products. |
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※ "This kit is using “Single Tube" |
| 1)
Simplicity |
| :
All reagents for cDNA and PCR reaction are in one tube. |
| Experimental errors of researchers
can be minimized and the protocol is also very simple. |
| 2)
High efficiency & specificity |
| :
It shows an excellent purity and no RNase or DNase contamination. |
| Efficient and specific reverse transcription reaction
is implemented by OptiScriptTMRT System, |
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and specific amplification is performed by i-StarTaqTM DNA polymerase. |
| 3)
Stability |
| :
Stability for long term storage is increased by adding special stabilization material. |
| 4)
Reproducibility |
| :
All necessary reagents for reaction are premixed to minimize the error |
| during pipetting process and ensure
the high reproducibility by the thorough Q.C.. |
|
|
ONE CYCLE |
| Reverse
transcription reaction |
45℃ /
30min |
| Inactivation of RTase |
94℃ /
5min |
|
3-STEP CYCLING |
| Denaturation
|
94℃ /
20-60sec. |
| Annealing |
45-68℃
/ 20-60sec. |
| Extension |
72℃
/ 1min/kb |
| Number
of cycles : 25-40
|
|
ONE CYCLE |
| Final
Extension |
72℃
/ 5min |
|
|
| - Transcription level and sequence of mRNA.
- Detection of antigen which is caused by the various disease.
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*
ONE-STEP RT-PCR Premix Solution
|
400㎕ |
1)
|
Oligo(dT)15 primer or random primer is not used for reverse transcription reaction.
Reverse transcription reaction and PCR reaction is implemented by gene specific primer. |
|
|
 Example of comparison experiment for gene expression amount |
ONE-STEP RT-PCR PreMix Kit is a product that all reagents for cDNA and PCR reaction are in one tube. This kit is suitable for the quantitative experiment
such as comparison experiment of gene expression amount because it can decrease the possibility of unexpected error conducted by researchers. |
 |
Fig.
Comparison experiment of Bcl-2 gene expression in various human bloodl cell line.
Extraction of RNA with easy-BLUEc from blood tumor cell line in human, and had a test of gene expression level from each cell line
with ONE-STEP RT-PCRTM. |
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| Sensitivity and specificity |
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ONE-STEP RT-PCR PreMix Kit selects OptiScriptTM RT System
and i-StarTaqTMDNA Polymerase for the high efficiency and specificity. |
| <
Element I : OptiScriptTM RT System > |
|
Fig.
RT PCR by AMV RT and OptiScriptTM
After extracting total RNA from human cancer cell lines (SNU 5) with easy-BLUETMTotal RNA Extraction Kit (Cat. No. 17061),
first strand of cDNA is synthesized by AMV Reverse Transcriptase (Cat.No. 27021) and Optiscript™ RT System respectively.
The solution containing synthesized cDNA is diluted up to proper concentration and the degrees of β-Actin(400bp) gene expression are compared. |
| <
Element II : i-StarTaqTM DNA Polymerase
> |
 |
Fig.
i-TaqTM Amplification of 4.5 kb gene cloned in the vector using i-Taq™ DNA polymerase and
i-StarTaq™ DNA polymerase respectively.
i-StarTaq™ DNA polymerase is more efficient than general Taq DNA polymerase to amplification of long size DNA.
8㎕ of PCR reaction mixture load on the 0.8% agarose gel.
Lane M, 1Kb Ladder DNA Marker;
Lane 1,2, i-TaqTM;
Lane 3,4, i-StarTaqTM
DNA Polymerase |
 |
Fig.
RT-PCR reaction by ONE-STEP RT-PCR PreMix Kit
Extraction of total RNA from human cancer cell lines(SNU 5) with easy-BLUETM Total RNA Extraction Kit (Cat. No. 17061),
and dilute the RNA for using ONE-STEP RT-PCR PreMix Kit to see the gene expression of β-Actin (400bp).
Lane M, 100bp Ladder DNA Marker; Lane
1, 1㎍ total RNA; Lane 2, 10ng total
RNA;
Lane 3, 1ng total RNA; Lane 4,
100pg total RNA; Lane 5, 10pg total RNA;
Lane 6, 1pg total RNA; Lane 7, Negative
control |
|
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| Example of use for disease detection |
ONE-STEP RT-PCR
PreMix Kit can be used to detect various disease due to the high efficiency and specificity. |
 |
Fig. Detection result of Newcastle virus with ONE-STEP RT-PCR PreMix
Total RNA is extracted from Newcastle virus with Viral Gene- spin™ Viral DNA/RNA Extraction Kit (Cat.No. 17151) and first strand cDNA synthesis and
PCR reaction is implemented by ONE-STEP RT-PCR PreMix Kit. In case of non-pathogenic sample, 379bp band is detected with RT-PCR and 204bp band
is detected with RT-PCR for the pathogenic sample.
Lane M, 100bp Ladder DNA Marker; Lane 1, 2, Vaccinated cell line; Lane
3, 4, 5, 6, 7, pathogenic cell line |
 |
Fig.
Detection result of Newcastle virus with ONE-STEP RT-PCR PreMix
Total RNA is extracted from Newcastle virus with Viral Gene- spin™ Viral DNA/RNA Extraction Kit (Cat.No. 17151)
and first strand cDNA synthesis and PCR reaction is implemented by ONE-STEP RT-PCR PreMix Kit.
Lane C, Negative control; Lane 1,
104 TCID50,;,Lane 2,
103 TCID50,; Lane 3,
102 TCID50;
Lane 4, 101 TCID50,;
Lane 5, 100 TCID50; Lane
6, 10-1 TCID50 |
 |
Fig.
Detection result photo of Newcastle disease virus from serially diluted allantoic fluid by ONE-STEP RT-PCR PreMix.
108.0EID50 /0.1㎖ allantoic fluid is serially diluted and total RNA is extracted with Viral Gene-spin™ (Cat. No.17151). First strand cDNA and PCR reaction
is implemented by ONE-STEP RT-PCR PreMix Kit
Lane M,
100bp Ladder DNA Marker; Lane 1, 10-2
diluted; Lane 2, 10-3 diluted; Lane
3, 10-4 diluted; Lane 4, 10-5
diluted; Lane 5, 10-6 diluted;Lane
6, 10-7 diluted; Lane 7, 10-8
diluted; Lane 8 : 10-9 diluted |
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| RT-PCR from various templates
|
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Fig. RT-PCR from various templates
Total RNA is extracted from virus and cells with using Viral Gene- spin™ Viral DNA/RNA Extraction Kit (Cat.No. 17151) and
easy-BLUE™ Total RNA Extraction Kit (Cat. No. 17061). First strand cDNA and PCR reaction is implemented by ONE-STEP RT-PCR PreMix Kit.
Lane M1, 100bp Ladder DNA Marker; Lane
M2, 1Kb Ladder DNA Marker;
Lane 1, Newcastle Disease Virus HN (120bp); Lane
2, Hog Cholera Virus NCR (421bp);
Lane 3, Infectious Bursal Disease Virus VP2 (450bp);
Lane 4, Bfl-1 (570bp), Bcl-2 family;
Lane 5,Human β-Actin (890bp); Lane 6,
hnRNP (900bp), alternative splicing factor
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