RT-PCR系列產品

RT-PCR & RT-qPCR & RT反轉錄酵素

基因的表達分析，通常從RNA起始。而RNA的多樣性和不穩定性，往往對下有實驗帶來挑戰。Zgenebio的RNA保存/提取產品使用極其方便，避免了繁瑣的液氮凍存、研磨，而獲得的RNA品質同傳統方法一樣好。基於我們專利的HiScript逆轉錄酶衍生的一系列產品，針對RT-PCR和RT-qPCR分別優化；更有簡便的一管式SuperMix提供。

Reverse Transcriptase 反轉錄酶

具有超強耐熱性和延伸效率反轉錄酶
HiScript II Reverse Transcriptase

訂購資訊:ZGVR201-01 / 02 2000U / 10000U

具有超強耐熱性和延伸效率的新一代反轉錄酶
HiScript III Reverse Transcriptase

訂購資訊:ZGVR302-01 / 10000U

HiScript III可將逆轉錄溫度提高至50~55℃，避免RNA複雜二級結構對cDNA合成的抑制

• 50℃半衰期超過240分鐘，55℃半衰期可達60分鐘
• 可合成創紀錄的20 kb全長cDNA
• 5分鐘即可合成長達10 kb的cDNA，適合快速反轉錄反應
• 更高的雜質耐受度
• 更高的檢測靈敏度，分子診斷首選反轉錄酶
產品描述
HiScript Reverse Transcriptase的基礎上，我們通過專利的體外分子進化技術，獲得了一個全新的反轉錄酶突變體-HiScript II Reverse Transcriptase。其攜帶的多個點突變使得其熱穩定性大幅提高，在50℃的半衰期超過240分鐘，並可在55℃長時間反應而保持穩定，保證了具有複雜二級結構模版的反轉錄成功率。同時，高溫下保持的高活性能夠帶來更高的cDNA產量。同時，HiScript II Reverse Transcriptase與模版的親和力 (affinity)以及行進性 (processivity)大幅提高，表現為極強的cDNA持續合成能力。HiScript II Reverse Transcriptase可從小鼠骨骼肌總RNA中反轉錄獲得創紀錄的20.0 kb的cDNA (圖2)，並且5分鐘內的合成長度可達10 kb，非常適合快速反轉錄反應。HiScript® II Reverse Transcriptase對於常見的反轉錄抑制劑具有更好的耐受度。這些抑制劑通常是RNA純化試劑的組分，或者是來源於樣品，在純化過程中會有不同程度的殘留，並在逆轉錄時被引入，如 guanidine、乙醇、木聚糖、EDTA、SDS、CTAB。與野生型的M-MLV (H-)以及其它反轉錄酶相比，HiScript II Reverse Transcriptase對雜質耐受度更高。這對於來源複雜的樣品 (如FFPE組織)，或者富含多糖多酚的植物樣品來說更為重要

圖1. 四種不同的逆轉錄酶分別在50℃和55℃與RT reaction mixture恆溫培育不同時間。剩餘的酶活用標準方法測定。 SS III: Superscript III。

圖2. 以1 μg HeLa細胞總RNA或小鼠骨骼肌細胞總RNA (for Nebulin)為模版，用HiScript® II 1st Strand cDNA Synthesis Kit (R211-01)進行逆轉錄。取1 μl cDNA為模版，用Vazyme LAmp® DNA Polymerase放大。 M. DL15000 Marker.

圖3. 以1 μg HeLa細胞總RNA為模版，Oligo dT23 VN為引子，分別用HiScript® II Reverse Transcriptase或Superscript® III Reverse Transcriptase (SSIII)在50℃進行逆轉錄，反應時間設置為5、15、30、45分鐘。取1 μl cDNA為模版，用LAmp® DNA Polymerase放大Fibrillin基因10.0 kb或6.1 kb片段。M. DL15000 Marker.

圖4. 以1 μg HeLa細胞總RNA為模版，分別用HiScript® II Reverse Transcriptase和T品牌逆轉錄酶進行逆轉錄反應，時間設置為5 min。取1 μl cDNA為模版，用LAmp® DNA Polymerase放大FIB 6100 bp和Pole 7117 bp片段。

圖5. 以1 μg- 1 pg HeLa細胞總RNA為模版，用HiScript® II Q RT SuperMix for qPCR (R222-01)做逆轉錄，取1 μl cDNA為模版，分別用AceQ® qPCR SYBR® Green Master Mix放大B2M基因 (A) 和EGFR基因 (B)。

One Step RT-PCR Kit

產品介紹：
One Step RT-PCR Kit專為以RNA為模版(如RNA病毒)的終點法PCR檢測而設計。使用基因專一引物(GSP)，逆轉錄和PCR反應在一管內完成，不需要額外的操作，大大提高了檢測速度，並降低了污染的風險。整合Reverse Transcriptase以及AceTaq DNA Polymerase的優越性能，配合經過優化的Buffer ，One Step RT-PCR Kit的檢測靈敏度可達到1 pg總RNA。試劑組以便捷的Master Mix形式提供。2 × Ace-Hi TM Mix包含優化的Buffer和dNTP； Enzyme Mix包含比例優化的Reverse Transcriptase、RNase inhibitor以及AceTaq DNA Polymerase。

◎方便快速: 使用單管單步驟RT-PCR系統
◎獨特配方: 使用MMLV RT 及Taq 酵素混合液 ( Enzyme Mix) 以及特殊reaction buffer , 效果穩定.
◎敏感度高: 附獨家配方Thermo-Enhancer, 使RT可在60℃反應, 可解RNA二級結構,提高RT-PCR產量及長度.

order info
ZGVP601-50 50RXN

RT-PCR of 220bp zebraf ish EF-1-α gene by RT-PCR kit.
0.5ng zebrafish total RNA was used for RT-PCR, RT step was performed at 50°C and 60°C respectively.
Lane 1 : RT-PCR by supplier A
Lane 2 : RT-PCR by supplier B with Enhancer
Lane 3 : RT-PCR by One RT-PCR kit with Thermo-Enhancer

One-Step RT-PCR Kit llI (SuperSAMscript III RTase/GenHot)

訂購資訊:ZGGMRP01-III 50RXN

The One-Step RT-PCR Kit llI provides one-tube, two-enzyme system for the reverse transcription and PCR amplification of a specific target RNA from either total RNA or mRNA. The system uses SuperSAMscript III reverse transcriptase for first-strand cDNA synthesis and GenHot DNA polymerase for PCR amplification. SuperSAMscript III RTase is a genetically engineered version of M-MLV RTase which lacks 3’ to 5’ exonucleolytic proofreading function and has a no RNase H activity as compared to AMV RTase, that is thermo stable (amplified DNA in 37~55 ℃ ) and is useful for full-length cDNA amplification(~15kb). GenHot Taq DNA polymerase in the kit is modified with specific monoclonal antibody and can eliminate amplification artifacts such as primer-dimer formation and mis-priming during pre-amplification stage.

Copies of RNA target

Figure 1. Sensitivity of One Step RT-PCR Kit lll.
Amplification of specific RNA from serial dilution of RNA templates with One-Step RT PCR Kit lll. RNA templates were prepared by in vitro transcription, and copies numbers were determined and calculated by spectrophotometer and gel phosphor screen imaging.
M: GM100 DNA Ladder
Template: E.coli total RNA

Two-Step SuperSAMscript III RT-PCR Kit

訂購資訊:ZGGMRP012-M3 50RXN

The Two-Step SuperSAMscript III RT-PCR Kit is provided with all components required to perform first-strand cDNA synthesis and second strand DNA amplification. SAMscript RTase is genetically engineered version of M-MLV RTase which is a frequent choice for cDNA synthesis because of its ease of use and low intrinsic RNase H activity. In the cDNA synthesis step, RNA is reverse transcribed by SuperSAMscript III RTase to produce its cDNA. For the subsequent amplification of the cDNA template, PCR Hot-Start Master Mix ll is provided. It can eliminate amplification artifacts such as primer-dimer formation and mis-priming during pre-amplification stage and thus may provide improved specificity. PCR Hot-Start Master Mix II can be used for effective amplification of DNA up to 15 kb in length.

Figure1. RT-PCR result of E.coli (DH5α) total RNA with amplification of second- strand cDNA fruK gene.
GeneMark Two-Step SuperSAMscript III RT-PCR Kit is used for E.coli (DH5α) total RNA reverse-transcription into cDNA and fruK gene PCR amplification.
M: GM100 DNA Ladder

Order info

產品	規格	貨號
HiScript llReverse Transcriptase	2000/10000 U	ZGVR201-01/02
HiScript1st Strand cDNA Synthesis Kit	50 rxn (20 μl/rxn)	ZGVR111-01
HiScript1st Strand cDNA Synthesis Kit	100 rxn (20 μl/rxn)	ZGVR111-02
HiScriptQ 1st Strand cDNA Synthesis Kit for Qpcr	200 rxn (10 μl/rxn)	ZGVR121-01
HiScriptQRT SuperMix for qPCR	200 rxn (10 μl/rxn)	ZGVR122-01
HiScript Q RT SuperMix for qPCR（+g DNA wiper）	200 rxn (10 μl/rxn)	ZGVR123-01
HiScript Q Select RT SuperMix for qPCR	200 rxn (10 μl/rxn)	ZGVR132-01
HiScript Q Select RT SuperMix for qPCR（+g DNA wiper）	200 rxn (10 μl/rxn)	ZGVR133-01
M-MLV Reverse Transcriptase	10000U	ZGVR011-01
M-MLV(H-) Reverse Transcriptase	10000 U	ZGVR021-01
RNase inhibitor	2000U 40U/ul	ZGGMRI-M007S
RNase H (E. coli )	250U (5U/μl)	ZGGMGD036S