BioRT逆转录扩增(RT-PCR)试剂盒说明书(一步法)

For research use only

b、按以下条件进行PCR反应

94℃ 3min

94℃ 30s

37℃-65℃ ** 30s

72℃ **** 45s-7min

72℃ 5min

注： * RT产物可增加至5 μl 。

** 退火温度根据引物Tm值调整，一般为Tm-5℃ 。Control引物退火温度为55℃。

*** 当实验样品RNA特别稀少时，可将循环数增加至40-45循环。

**** 延伸时间根据PCR产物大小确定，一般1kb/min 。Control引物延伸时间45s。

25-35循环 ***

c、反应结束后，取3-5 μl PCR产物进行琼脂糖凝胶电泳，确认PCR反应产物。如果此PCR产物需用于以后实验，应将PCR产物冷冻保存。

RNA control反应时，退火温度55度，30个循环，扩增片段大小为500bp。

使用提示

1. RNA模板可以采用总RNA或mRNA，建议使用Biozol(BSC51M1)制备高质量RNA；

2. RT实验应避免RNase污染，可采用以下措施：

1） 因人的皮肤表面和唾液都有RNase，因此实验中应戴一次性手套和口罩；

2） RT实验应使用专门的仪器和耗材，建议在专门区域操作RNA；

3） RT实验相关耗材应使用干热灭菌（180℃，60分钟）或用0.1％ DEPC(焦碳酸二乙酯)水溶液在37℃处理12小时后在121℃高压灭菌30分钟；

3. AMV逆转录酶，DNA聚合酶和RNase抑制剂在取用之前应离心后再吸取，吸取时动作要慢，使用后应尽快放回-20℃；

4. dNTP应避免反复冻融以免失效；

5. 引物的选择可根据具体情况，Oligo-dT适用于具有PolyA尾的RNA(一般是真核生物的mRNA)，Random Hexamer Primer适用于所有RNA(包括mRNA,rRNA,tRNA等)，尤其适用于有复杂二级结构的RNA，特异性引物适用于已知模板序列的RNA；

6. PCR反应中MgCl2浓度可依据不同条件进行调整，当目的片段长度大于2kb时，我们建议增加MgCl2浓度，以0.5mM间隔梯度增加。

7. PCR引物的设计的好坏直接影响到PC反应的结果，设计PCR引物考虑多种因素，如GC含量，引物长度，引物位置等因素，因此我们建议采用优秀的引物设计软件来设计，如Primer Premier 5.0等。

RT-PCR实验必需用品

仪器和耗材

离心机

微量移液器

RNase free 1.5ml离心管

水浴装置或金属浴装置

电泳及UV装置

PCR管

移液器吸头

参考文献

试剂

DEPC(焦碳酸二乙酯)

ddH2O

电泳缓冲液

上样缓冲液

DNA Marker

1. Houts, G.E., Miyagi, M., Ellis, C., Beard, D., and Beard, J.W. (1979) .

29(2):517-522.

2. Guide to Molecular Cloning Techniques. Methods in Enzymology, Volume 152. pp

316-325. Edited by Shelby Berger and Alan R. Kimmel. Academic Press, Inc.

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For research use only

BioRT Two Step RT-PCR Kit

BioRT逆转录扩增(RT-PCR)试剂盒说明书(两步法)

Cat No.: BSB05M1

TECHNICAL SUPPORT:

For technical support, please dial phone number ：

0086-571-87774567-5278, 5211 or 800-857-1279

************************.e:

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For research use only

产品说明

RT-PCR是指利用逆转录酶将RNA逆转录(RT)成cDNA(Complementary DNA)，然后以cDNA为模板，通过聚合酶链式反应(PCR)扩增目的片段的技术。RT-PCR技术可用于检测细胞和组织中基因表达水平，克隆特定基因的cDNA序列和检测RNA病毒。

BioRT逆转录扩增(RT-PCR)试剂盒采用美国先进技术生产的高质量逆转录酶(AMV酶)和高保真的Taq mix DNA聚合酶，AMV逆转录酶可逆转录得到高产量的cDNA，并可逆转录长达12kb的cDNA，同时AMV逆转录酶有较高的热稳定性，其反应温度可高达60℃，可以逆转录具有复杂二级结构的RNA模板， Taq mix DNA聚合酶同时具有高保真，高灵敏，高延伸速度等特点，可合成长达6Kb的PCR产物，两种酶的配合使用保证了RT-PCR反应的顺利完成。

本试剂盒采用两步法进行RT-PCR，即RT和PCR分别在两管中进行，同一次逆转录的cDNA能同时检测多个基因，使得您有限的RNA能发挥最大的作用。

RT-PCR原理

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For research use only

Components

(100 rxns)

AMV Reverse Transcriptase(5U/μl)

5 x RT Buffer

dNTP Mixture(10mM)

RNase inhibitor(40U/μl)

Oligo-dT(18)

Random Primer

RNA control (freeze dryness)

RNase free H2O

52 μl

500 μl

200 μl

52 μl

52 μl

52 μl

Add 20ul*

1 ml×2

Taq mix DNA polymerase(5U/μl) 52 μl

10 x PCR Buffer (include 15mM

Mg2+)

500 μl

MgCl2(25mM)

200 μl

RNA control S primer (5μM)

RNA control A primer (5μM)

Store at -20℃

20 μl

20 μl

Protocol

1. RT reaction

a、Reaction setup for cDNA synthesis

5 x RT Buffer 2 μl

dNTP Mixture (10Mm) 1 μl

Oligo-dT

or Random Hexamer Primer** 0.5 μl

or special downstream primer

RNase inhibitor (40U/ul) 0.5 μl

AMV Reverse Transcriptase (5U/ul) 0.5 μl

RNA Control or Sample RNA* x μl

RNase free H2O 5.5-x μl

total 10 μl

Notes

*Please add 20ul RNase free H2O before using RNA Control and dissolve sufficiently. Add

2ul/test when you do RNA valume can be add up to 5.5ul (≤1ug ).

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For research use only

b、RT reaction condition

(room temprature，10min) **

42-60℃ ***，45min

95℃ ****，5min

ice bath，5min

cDNA for PCR reaction.

Notes

** If using random Hexamer Primer, holding at room temprature for 10 minutes.

*** Reaction temperature can be elevated for RNA template with second structure，lower

than 60℃, or holding at 70℃ for 10 minutes after adding RNA template and primers,then

holding on ice for 5 minutes.

**** Heat to 95℃, let AMV Reverse Transcriptase denature.

2. PCR protocol

a、Reaction setup for PCR

10 x PCR Buffer (include 15mM

Mg2+) 2.5 μl

dNTP Mixture (10mM) 0.5 μl

Upstream primer (5 μM) 0.5 μl

Downstream primer (5 μM) 0.5 μl

Taq mix DNA polymerase 0.5 μl

cDNA* 2.5 μl

ddH2O 18 μl

total 25 ul

b、PCR reaction condition

94℃ 3min

94℃ 30s

37℃-65℃ ** 30s

25-35cycles ***

72℃ **** 45s-7min

72℃ 5min

Note:

* RT products can up to 5.0 μl.

** 5℃ lower than Tm value of primers；Control primer is 55℃.

*** Add to 40-45 cycles when detecting rare RNA templates .

**** usually 1kb/min . Control primer is 45s.

c、Analyze 3 - 5μl of the reaction products by agarose gel

electrophoresis.

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For research use only

Using Tips

1.

Total RNA or mRNA can be used as RNA template，and we suggest using

Biozol(BSC51M1)to isolate high quality RNA；

2.

RNase contamination should be avoided and follow the measures:

1） Ware one time gloves and respirator because of the RNases in saliva

and skin;

2） Use special instruments and consumables and handle in specific areas;

3） Consumables should be treated at 180℃ for 60min or 37℃ for 12 hours

with 0.1% DEPC H2O followed by sterilized at 121℃ for 30min;

3.

AMV reverse transcriptase, Taq polymerase and RNase inhibitor should be

slowly pipetted after centrifuging, and put back at -20℃ later;

4.

Avoid frequently freezing and thawing dNTP;

5.

Specific primers should be used and concentration of primers should be

optimized and We suggest 0.4μM as a starting point for optimizing; Oligo-dT and

Random primers are not suitable for this kit;

6.

Concentration of MgCl2

can be optimized and increasing 0.5mM/time is

suggested when the length of target template is longer than 2kb;

7.

Quality of primers affect the performance of BioRT One Step RT-PCR Kit;

Factors such as GC percentage, length of primers and site of primers should be

considered and we suggest primers be designed using special soft wares.

Required materials for RT-PCR

Instruments and consumables Reagents

Centrifuge DEPC

Pipettes ddH2O

RNase free 1.5ml tubes Electrophoresis Buffer

Water bath instruments Loading Buffer

Gel electrophoresis instruments DNA Marker

PCR tubes

Tips

Reference

1. Houts, G.E., Miyagi, M., Ellis, C., Beard, D., and Beard, J.W. (1979) .

29(2):517-522.

2.

Guide to Molecular Cloning Techniques. Methods in Enzymology, Volume

152. pp 316-325. Edited by Shelby Berger and Alan R. Kimmel.

Academic Press, Inc.

5 / 8

For research use only

Description

RT PCR is a useful technique for the investigation of gene expression, viral

load, pathogen detection, and numerous other applications. When analyzing

gene expression or viral load, the RNA of interest first needs to be reverse

transcribed into cDNA. The subsequent PCR can be performed separately

(two-step RT-PCR).

BioRT Two Step RT-PCR Kit is designed for two step RT-PCR of RNA

samples from various sources. The kit includes all the necessary reagents for

cDNA synthesis and subsequent PCR. Either total RNA, messenger RNA, viral

RNA or in vitro transcribed RNA can be used as a template for reverse

transcription. The kit includes both random primers and oligo(dT18) primers.

The user can choose either of these or alternatively use gene specific primers.

The reverse transcriptase in the BioRT Two Step RT-PCR Kit is AMV, which

provides up to 60℃ RT temperature and provides higher sensitivity and higher

yield to cDNA’s synthesis and PCR. The performance of the PCR step is based

on a high fidelity Taq mix DNA polymerase. The kit can synthesis up to 12kb

cDNA and 6kb DNA. The reaction buffer is optimized for a kind of reaction

system (10ul for RT and 25ul for PCR).

RT-PCR Principle

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For research use only

试剂盒组成 (100次使用量)

AMV Reverse Transcriptase(5U/μl)

5 x RT Buffer

dNTP Mixture(10mM)

RNase inhibitor(40U/μl)

Oligo-dT(18)

Random Primer

RNA control ( 冷冻干燥 )

RNase free H2O

Taq mix DNA polymerase(5U/μl)

10 x PCR Buffer（含15mM Mg2+）

MgCl2(25mM)

RNA control S primer(5μM)

RNA control A primer(5μM)

保存 -20℃

52 μl

500 μl

200 μl

52 μl

52 μl

52 μl

加20 μl*

1 ml×2

52 μl

500 μl

200 μl

20 μl

20 μl

实验操作Protocol

1. RT反应

a、按以下条件配制RT反应液

5 x RT Buffer 2 μl

dNTP Mixture(10mM) 1 μl

Oligo-dT

或 Random Hexamer Primer ** 0.5 μl

或特异性下游引物

RNase inhibitor(40U/ul) 0.5 μl

AMV Reverse Transcriptase 0.5 μl

RNA Control或实验样品 RNA * x μl

RNase free H2O 5.5-x μl

总体积 10 μl

注：* RNA Control使用前加20ul RNase free H2O水充分溶解，每次反应加2ul；实验样品RNA体积根据浓度确定，总量小于等于1ug总RNA；当实验样品RNA的表达数量较少时，可加至5.5ul，同时在反应体系中相应地减少RNase free H2O 的量。

b、按以下条件进行逆转录反应

( 室温 10min ) **

42-60℃ *** 45min

95℃ **** 5min

冰浴 5min

注 ：** 对于随机六聚体引物，在室温温育10分钟后，再进行逆转录反应。

*** 对于有复杂二级结构的RNA模板，逆转录温度可适当提高，不可高于60℃；也可以加模板和引物后70℃10min，再冰浴5min后继续后续实验；逆转录长链RNA （> 2kb）时，建议在42℃左右进行。

**** 95℃加热使AMV Reverse Transcriptase失活并阻止其与DNA结合。

2. PCR反应

a、 按以下条件配制PCR反应液

10 x PCR Buffer （含Mg2+） 2.5 μl

dNTP Mixture (10mM) 0.5 μl

上游特异性引物 (5 μM) 0.5 μl

下游特异性引物 (5 μM) 0.5 μl

Taq mix DNA polymerase 0.5 μl

RT产物 * 2.5 μl

ddH2O 18 μl

总体积 25 μl

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