invitrog逆转录试剂盒说明书

ThermoScript RT-PCR System

Catalog nos. (25 reactions): 11146-024

11146-057 (w/ Platinum Taq DNA polymerase)

Description

The ThermoScript RT-PCR System is designed for the sensitive and reproducible detection and analysis of RNA molecules in a two-step process. ThermoScript RT, an avian reverse transcriptase with reduced RNase H activity, is engineered to have higher thermal stability,

produce higher yields of cDNA, and produce more full-length cDNA transcripts than AMV RT. cDNA synthesis is performed in the first step using either total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers or a gene-specific primer, at 50-65°C. In the second step, PCR is performed in a separate tube using primers specific for the gene of interest. RNA targets from 100 bp to >12 kb can be detected with this system, using 10 pg to 5 Yg of total RNA. PCR is carried out with Platinum Taq DNA Polymerase or Platinum Taq DNA Polymerase High Fidelity. Platinum Taq DNA Polymerase High Fidelity is suitable for templates from 100 bp to >12 kb. Platinum Taq DNA polymerase (1) provides automatic hot-start conditions for increased specificity up to 3 kb.

Catalog nos. (100 reactions): 11146-016

11146-032 (w/ Platinum Taq DNA polymerase)

11146-040 (w/ Platinum Taq DNA polymerase High Fidelity) Store at -20°C (stability can be extended by storing at -70°C)

Reagents are provided for 25 or 100 cDNA synthesis reactions of 20 μl each and 25 or 100 amplification reactions of 50 μl each.

Catalog numbers 11146-057 (25 rxns) and 11146-032 (100 rxns) include the following, in addition to the components to the left:

ThermoScript RT (15 U/Yl) 25 μl 100 μl Platinum Taq DNA polymerase (5 U/Yl) 100 units 250 units

5X cDNA Synthesis Buffer* 500 μl 500 μl 10X PCR buffer Minus Mg 1.0 ml 1.0 ml 0.1 M DTT 250 μl 250 μl 50 mM MgCl2 1.0 ml 1.0 ml 10 mM dNTP Mix 100 μl 2 × 250 μl

Catalog number 11146-040 (100 rxns) includes the following, in RNaseOUT (40 U/Yl) 25 μl 100 μl

addition to the components to the left: Oligo (dT)20 (50 YM) 25 μl 100 μl

Random Hexamers (50 ng/Yl) 50 μl 250 μl

Platinum Taq DNA Polymerase DEPC-Treated Water 1.25 ml 1.25 ml

High Fidelity (5 U/μl) 100 units E. coli RNase H (2 U/Yl) 50 μl 2 × 50 μl

10X High Fidelity PCR Buffer 1.0 ml *250 mM Tris acetate (pH 8.4), 375 mM potassium acetate, 40 mM

50 mM MgSO4 1.0 ml magnesium acetate, stabilizer

Quality Control

The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available on our website at

.

Summary of Procedure

*If less than 1 ng of RNA is used, reduce the amount of ThermoScript RT in the reaction to 0.5 μl. andom hexame 20 GSP

50-65°25°C, 10 min ↓ 50-60° ↓

85°C, 5 min

↓ Add 1 μl RNase H r37°C, 20 min ↓ Remove 2 μl aliquot for PCR

Part no. 11146.pps MAN0000941 Rev. date: 11 Jun 2010

For technical support, email [email protected]. For country-specific contact information, visit www.invitrogen.com.

Important Parameters to Consider

RNA

• High quality intact RNA is essential for successful

full-length cDNA synthesis and successful long RT-PCR.

• RNA should be devoid of any RNase contamination

and aseptic conditions should be maintained. • Recommended methods of total RNA isolation

include the Micro-to-Midi Total RNA Purification System (Catalog no. 12183-018) and TRIzol Reagent (Catalog no. 15596-026) (2, 3). Oligo(dT)-selection for poly(A)+ RNA is typically not necessary, although incorporating this step may improve the yield of specific cDNAs.

cDNA Synthesis Primers

• Oligo(dT)20 (50 pmoles/reaction) is recommended for

priming polyadenylated RNA. Use of Oligo(dT)20 allows the detection of multiple transcripts from a single first-strand reaction.

• Random hexamers (50-250 ng/reaction) are efficient

primers for the detection of multiple short RT-PCR targets. Use of more than 50-100 ng primer/Yg of RNA can increase the yield of short products but may inhibit detection of long targets (>3kb) or rare

transcripts. If random hexamers are used, the first-strand reaction must be incubated at 25°to extend the primers prior to increasing the reaction temperature for synthesis.

• Gene-specific primers (GSP) should be used at 10 to

20 pmol/reaction. Specificity of priming may be improved by optimizing annealing/reaction temperature.

• Treatment of cDNA with RNase H to remove the

complementary RNA prior to PCR is optional. RNase H digestion will improve the RT-PCR signal of many targets and is required for the efficient and consistent amplification of long RT-PCR templates. cDNA Synthesis Reaction

• Denaturation of the RNA template and primer by

incubating at 65°can be reverse transcribed efficiently without this step. However, heating the RNA in the absence of reaction buffer and enzyme prior to cDNA synthesis can remove secondary structure that may impede full-length cDNA synthesis.

• ThermoScript RT can be used at 50-65°C. We

recommend incubation at 50-55°C for most RT-PCR targets. However, incubation at 50-60°C for oligo(dT) and 50-65°C for gene-specific primers can be

employed to reduce secondary structure or to improve specificity.

• Most targets can be amplified after only a 30-min

incubation for the first-strand reaction. Rare RNAs, long transcripts, or targets at the 5′ end of long

transcripts benefit from longer incubation times (50-60 min).

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PCR Primers

• A final primer concentration of 0.2 - 0.4 μM for each

primer is generally optimal; however, a primer titration is recommended for best results.

• Design primers that anneal to sequence in exons on

both sides of an intron or exon/exon boundary of the mRNA to allow differentiation between amplification of cDNA and potential contaminating genomic DNA. • Primers should not be self-complementary or

complementary to each other at the 3′ ends. PCR Reactions

• Most targets will be efficiently amplified using 2 Yl or

less of the cDNA synthesis reaction.

• The optimum magnesium concentration varies from

1.5 to 3 mM. Generally, 1.82 mM magnesium chloride for Platinum Taq DNA polymerase and 2.32 mM magnesium sulfate for Platinum Taq DNA

Polymerase High Fidelity is effective for most primer sets. However, titration of the magnesium

concentration is recommended for the best result. Each Yl of the cDNA synthesis reaction adds 0.16 mM to the final magnesium concentration in a 50-μl PCR reaction.

• Assemble the PCR reactions on ice, transfer them to a

pre-heated thermal cycler (85-95°C) and immediately start the PCR amplification program.

• The annealing temperature should be 10°C below the

melting temperature of the primers used.

• The optimum extension temperature for Platinum

Taq DNA Polymerase High Fidelity is 68°C. The extension time varies with the size of the amplicon (approximately 1 min per 1 kb of amplicon).

cDNA Synthesis

1. In a 0.2- or 0.5-ml tube, combine primer (50 μM Oligo(dT)20, 50 ng/μl random primer or 10 μM gene-specific primer), RNA, and dNTP mix and adjust volume to 12 Yl with DEPC-treated water. Primer 1 Yl RNA (10 pg -5 Yg) x Yl 10 mM dNTP Mix 2 Yl DEPC-treated water to 12 Yl

2. Denature RNA and primer by incubating at 65°C for 5 min and then place on ice (optional).

3. Vortex the 5X cDNA Synthesis Buffer for 5 s just prior to 4. Prepare a master reaction mix on ice and vortex gently. 5x cDNA Synthesis Buffer 4 Yl 40 Yl 0.1 M DTT 1 Yl 10 Yl

RNaseOUT

(40 U/Yl) 1 Yl 10 Yl DEPC-treated water 1 Yl 10 Yl ThermoScript RT (15 units/Yl)1 Yl* 10 Yl* *NOTE: If less than 1 ng of template RNA is used, reduce the amount of ThermoScript RT in the reaction to 0.5 μl/reaction (5 μl/10 reactions). Increase the amount of DEPC-treated water in the master reaction mix to 1.5 μl/reaction (15 μl/10 reactions).

5. Pipet 8 Yl of master reaction mix into each reaction tube on ice.

6. Transfer the sample to a thermal cycler preheated to the appropriate cDNA synthesis temperature and incubate as follows.

Oligo(dT)20 primed: 30-60 min at 50°C (or 50-60°C) Gene-specific primed 30-60 min at 50°C (or 50-65°C) Random-hexamer primed: 25°C for 10 min, followed by 20-50 min at 50°C (or 50-65°C)

7. Terminate the reaction by incubating at 85°C for 5 min. 8. Add 1 Yl of RNase H and incubate at 37°C for 20 min (optional).

9. cDNA synthesis reactions can be stored at -20°C or used for PCR immediately. PCR with Platinum Taq DNA Polymerase High Fidelity Use only 10% of the cDNA synthesis reaction (2 Yl) for PCR. Use of 2 μl of 50 mM MgSO4 and 2 Yl of cDNA (0.32 mM magnesium in a 50-Yl PCR) results in a final concentration of 2.32 mM magnesium, which is effective for most primer sets. However, titration of the magnesium concentration with the provided 50 mM MgSO4 is recommended for best results. 1. Add the following to a 0.2- or 0.5-ml, thin-walled, PCR tube: 10X High Fidelity PCR Buffer 5 Yl 50 Yl

50 mM MgSO4 2 μl 20 μl 10 mM dNTP Mix 1 Yl 10 Yl 10 YM sense primer 1 Yl 10 Yl 10 YM antisense primer 1 Yl 10 Yl

Platinum

Taq High Fidelity 0.2 Yl 2 Yl cDNA (from cDNA synthesis reaction) 2 Yl 20 Yl Final volume 50 Yl 500 μl

Page 3 of 4

2. Mix gently and overlay with silicone oil or mineral oil if the

thermal cycler lacks a heated lid.

3. Incubate at 94°C for 2 min, then perform 20 to 40 cycles of PCR with optimized conditions for your sample (1 min/kb extension time at 68°C).

4. Analyze 10 Yl of the amplified sample by agarose gel electrophoresis. PCR with Platinum Taq DNA Polymerase

Use only 10% of the cDNA synthesis reaction (2 Yl) for PCR. Use of 50 mM MgCl2 and 2 Yl of cDNA will result in a final magnesium concentration of 1.82 mM, which is adequate for most primers and targets. However, titration of magnesium concentration is recommended for best results.

1. Add the following to a 0.2- or 0.5-ml, thin-walled, PCR tube: 10X PCR Buffer Minus Mg 5 Yl 50 Yl 50 mM MgCl2 1.5 Yl 15 Yl 10 mM dNTP Mix 1 Yl 10 Yl 10 YM sense primer 1 Yl 10 Yl 10 YM antisense primer 1 Yl 10 Yl

Platinum

Taq DNA polymerase 0.4 Yl 4 Yl (5 U /Yl)

cDNA (from cDNA synthesis reaction) 2 Yl 20 Yl Final volume 50 Yl 500 μl 2. Mix gently and overlay with silicone oil or mineral oil if the thermal cycler lacks a heated lid.

3. Incubate at 94°C for 2 min, then perform 20 to 40 cycles of PCR with optimized conditions for your sample (1 min/kb extension time at 68-72°C)

4. Analyze 10 Yl of the amplified sample by agarose gel electrophoresis. Control Reactions

An RT-PCR Primer and Control Set is available separately for monitoring the performance of the system (Cat. Number 10929-016).

1. Use 1 ng of the Control RNA in the cDNA Synthesis Reaction. 2. Perform the PCR using Platinum Taq DNA Polymerase, as described above.

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Troubleshooting Guide

Problem

No RT-PCR product

Possible cause

No cDNA synthesis (temperature too high) Incomplete synthesis of target cDNA

(secondary structure of RNA blocks synthesis) RNase contamination

Concentration of template RNA in reaction is too low

RNA has been damaged or degraded RT inhibitors are present in RNA

Possible solution

For the cDNA synthesis step, incubate at 45-50°C. For the cDNA synthesis step, incubate at 50-70°C. For long mRNAs, increase cDNA synthesis incubation time (up to 50 min)

Maintain aseptic conditions; add RNaseOUT (RNase inhibitor).

Increase the concentration of template RNA; use 1-5 μg of total RNA or reduce the volume of ThermoScript RT used in the reaction. Replace RNA.

Remove inhibitors in the RNA preparation by an additional 70% ethanol wash after ethanol precipation.

Note: Inhibitors of RT include SDS, EDTA, guanidinium chloride, formamide, sodium

phosphate and spermidine (4).

Low yield/low specificity in PCR

Unexpected bands after electrophoresis

Cycle number is too low Reaction conditions not optimal

RNA contamination with genomic DNA

Increase cycle number.

Optimize magnesium concentration. Optimize the primer concentration

Optimize the annealing temperature and extension time.

Increase temperature of RT reaction to 50-60°C. Pre-treat RNA with DNase I.

Redesign PCR primers to anneal to sequence in exons on both sides of an intron in the target gene.

References

1. 2. 3. 4.

Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus 19, 46. Chomczynski, P and Sacchi, N. (1987) Anal. Biochem. 162, 156.

Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry 18, 5294. Gerard, G.F (1994). Focus 16, 102.

Limited Use Label License No. 1: Thermostable Polymerases (applies to 11146-057, 11146-032, and 11146-040)

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224, 5,618,711, and 6,127,155. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Limited Use Label License No. 4: Products for PCR that include no rights to perform PCR (applies to 11146-016 and 11146-024)

This product is compatible for use in the Polymerase Chain Reaction (PCR) process claimed in patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No license under these patents is conveyed expressly, by implication, by estoppel or otherwise to the purchaser by the purchase of this product. A license to use these patented processes for certain research and development activities

accompanies the purchase of certain reagents of Applied Biosystems and other licensed suppliers when used in conjunction with an authorized thermal cycler, or is available from Applied Biosystems. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

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The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the

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Limited Use Label License No: 14: Direct Inhibition by Anti-Polymerase Antibodies (applies to 11146-057, 11146-032, and 11146-040)

Licensed to Life Technologies Corporation, under U.S. Patent Nos. 5,338,671; 5,587,287; and foreign equivalents for use in research only

Limited Use Label License No. 17: AFLP Products (applies to 11146-040)

The AFLP technique is covered by patents or patented applications owned by Keygene n.v and this product is sold under license from Keygene n.v. This kit may be used for research purposes only. For use of this kit in plant breeding, contact the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500. The use of this kit for any other purpose, including but not limited to the use for clinical, diagnostic, and/or therapeutic purposes; or for providing services to third parties, requires a license from Keygene n.v., P.O. Box 216, 6700 AE Wageningen, The Netherlands.

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