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Reverse Transcription

AMV Reverse Transcriptase

Product information

Features

• Optimum activity at 45-50°C.
• Highly thermostable, can be used at temperatures up to 60°C.
• Efficient synthesis of first strand cDNA up to 13 kb.
• Incorporates modified nucleotides.

Applications

• First strand cDNA synthesis for RT-PCR and real-time RT-PCR
• Reverse transcription at elevated temperatures to reduce the effects of secondary structure.
• Synthesis of cDNA for cloning and expression.
• Synthesis of labeled cDNA probes.
• Analysis of RNA by primer extension (1).

Description

AMV Reverse Transcriptase (RT) is a recombinant Avian Myeloblastosis Virus reverse transcriptase expressed in E.coli. AMV RT is a heterodimer composed of nonidentical alpha and beta subunits. It possesses multiple enzymatic activities including RNA- and DNA-directed DNA polymerase, DNA-RNA unwinding activity, a sequence-specific Mn²⁺-dependent endonuclease and ribonuclease H. AMV RT maintains activity over a wide temperature range (45-60°C), which makes it an ideal tool for reverse transcription of RNAs having a high degree of secondary structure.

Source

E.coli cells with a cloned pol gene of AMV.

Definition of Activity Unit

One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 min at 37°C.

Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 8 mM MgCl₂, 30 mM KCl, 1 mM DTT, 0.5 mM dTTP, 0.4 MBq/ml [³H]-dTTP, 0.4 mM polyA·oligo(dT)_12-18.

Storage Buffer

The enzyme is supplied in:
200 mM potassium phosphate, 2 mM DTT, 0.2% (v/v) Triton X-100 and 50% (v/v) glycerol (pH 7.2).

5X AMV RT Buffer

250 mM Tris-HCl (pH 8.5 at 25°C), 40 mM MgCl₂, 150 mM KCl, 5 mM DTT.

Quality Control

The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate. Sodium pyrophosphate reduces AMV RT activity, however at a higher than 4 mM concentration increases the yield of full-length cDNA (2).
• Inactivated by heating at 85°C for 5 min.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Guidelines to Avoid RNase Contamination

RNA purity and integrity is essential for synthesis of full-length cDNA, which results in high quality RT-PCR products. Therefore, RNase contamination is always a concern when working with RNA. The RNA quality can be affected by RNase A, which is a highly stable contaminant of any laboratory environment. All components of the kit have been rigorously tested to ensure that they are RNase free. To prevent contamination both the laboratory environment and all prepared solutions must be free of RNases.

• DEPC-treat all tubes and pipette tips to be used in the cDNA synthesis or use certified nuclease-free labware.
• Use pipettes dedicated for RNA work.
• Wear gloves when handling RNA and all reagents, as skin is an common source of RNases. Change gloves frequently.
• Use certified reagents, including high quality water (e.g., nuclease-free or DEPC-treated Water).
• Use an RNase inhibitor, such as RiboLock™ RNase Inhibitor, to protect template RNA.
• Always assess the integrity of RNA prior to cDNA synthesis. For example, if sharp bands of both the human 18S rRNA (runs at approx. 1.9 kb) and the 28S rRNA (runs at approx. 5 kb) are formed during denaturing agarose gel electrophoresis of total RNA, the mRNA in the sample is considered to be intact.

First Strand cDNA Synthesis

Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors and contamination, prepare a RT master mix by adding all reaction components except RNA into one vial. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Sample template RNA into individual tubes and keep on ice. Aliquote the prepared master mix into tubes with RNA. Mix and briefly centrifuge all components after thawing, keep on ice.

1. Add into sterile, nuclease-free tube on ice in the order given:
   

   Template RNA	Total RNA or Poly(A) RNA or Specific RNA	10 ng – 5 µg 1 ng-0.5 µg 0.01 pg – 0.5 µg
   Primer	Oligo(dT)18 primer (#SO131) or Random hexamer primer (#SO142) or Gene-specific	0.5 µg (100 pmol) 0.2 µg (100 pmol) 15-20 pmol
   Water, nuclease free (#R0581)		to 13 µl
   Total volume		13 µl

2. Optional: If RNA template is GC-rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min, chill on ice, briefly centrifuge and place on ice.
3. Add in the indicated order:

   5X AMV RT Buffer	4 µl
   dNTP Mix, 10 mM each (#R0191)	2 µl (1 mM final concentration)
   RiboLock™ RNase Inhibitor (#EO0381)	0.5 µl (20 u)
   AMV Reverse Transcriptase	0.5 µl (10 u)
   Total volume	20 µl

4. Mix gently and briefly centrifuge.
5. If oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 min at 50°C.
   If random hexamer primer is used, incubate 10 min at 25°C followed by 60 min at 50°C. For reverse transcription of GC-rich RNA reaction temperature can be increased up to 60°C.
6. Terminate the reaction by heating at 85°C for 5 min.

Do not heat inactivate enzyme prior to analysis of long cDNA to avoid cleavage.

The reverse transcription reaction product can be directly used in PCR, qPCR or second strand cDNA synthesis or stored at -20°C for up to one week. For longer storage -70°C is recommended. Avoid freezing/thawing of cDNA.
Use 2 µl of the reaction mix to perform PCR in 50 µl reaction volume.

Synthesis of cDNA Probes with High Specific Radioactivity and Non-radioactively Labeled cDNA

I. Synthesis of cDNA probes with high specific radioactivity

Mix and briefly centrifuge all components after thawing, keep on ice.

1. Add into sterile, nuclease-free tube on ice in the order given:

   Template RNA	Total RNA or	10 ng - 5 µg
   	Poly(A) RNA or	1 ng - 0.5 µg
   	Specific RNA	0.01 pg - 0.5 µg
   Primers	Oligo(dT)18 (#SO131) or	0.5 µg (100 pmol)
   	Random Hexamer (#SO142) or	0.2 µg (100 pmol)
   	Gene-specific	15-20 pmol
   Water nuclease free (#R0581)		to 9 µl
   Total volume		9 µl

2. Optional: if RNA template is GC-rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min, chill on ice, briefly centrifuge and place on ice.
3. Add into the same tube in the indicated order:

   5X AMV RT buffer	4 µl
   RiboLock™ RNase Inhibitor (#EO0381)	0.5 µl (20 u)
   dGTP, dCTP, dTTP mix, 10 mM each	1 µl
   0.1 mM dATP	4 µl
   [alpha-32P]-dATP, 3000 Ci/mmol	1 µl
   AMV Reverse Transcriptase	0.5 µl (10 u)
   Total volume	20 µl

4. Mix gently and centrifuge to collect all drops.
5. If Oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 min at 50°C. If random hexamer primer is used, incubate 10 min at 25°C followed by 60 min at 50°C. For reverse transcription of GC-rich RNA reaction temperature can be increased up to 60°C.
6. Terminate the reaction by heating at 85°C for 5 min.
   Optional. Hydrolyze RNA by the addition of equal volume (25 µl) of 0.6 M NaOH and incubation at 85°C for 5 min.
7. Remove unincorporated dNTPs by spin column (e.g. GeneJET PCR purification Kit, #K0701) or chromatography on a Sephadex® G-50 column.
8. Expect specific radioactivity of >10⁷ dpm/µg.

Note

• To achieve higher specific activities (over 10⁸ dpm/µg), use up to 100 µCi of [alpha-³²P]-dATP in the labeling mixture. To keep the total reaction volume of 20 µl, vacuum-evaporate 10 µl of [alpha-³²P]-dATP (10 mCi/ml) to 1 µl in a separate tube.

II. Synthesis of non-radioactively labeled cDNA

The protocol above can be used for synthesis of non-radioactive labeled cDNA using biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP or aminoallyl-dUTP:

• normal dTTP is subsituted with labeled-dUTP at a molar ratio of 1:3 - 1:4,
• reaction time is prolonged to 2-6 hours.

Removal of Genomic DNA from RNA Preparations

1. Add to an RNase-free tube:
   

   RNA	1 µg
   10X reaction buffer with MgCl2	1 µl
   DNase I, RNase-free	1 µl (1 u)
   DEPC-treated Water	to 10 µl
   Total volume	10 µl

2. Incubate 30 min at 37°C.
3. Add 1 µl of 50 mM EDTA and incubate 10 min at 65°C. RNA hydrolyzes if heated in the absence of a chelating agent (1).
4. Use the prepared RNA as a template for reverse transcriptase.

Note

• Do not use more than 1 u of DNase I, RNase-free per µg of RNA.
• Reaction mixture can be scaled up for larger amounts of RNA. The recommended final concentration of RNA is 0.1-0.2 µg/µl.
• RiboLock™ RNase Inhibitor (#EO0381), typically at 1 u/µl, can also be included in the reaction mixture to inactivate type A RNases potentially present in the initial RNA solution

Reference

1. Wiame, I., et al., Irreversible heat inactivation of DNase I without RNA degradation, BioTechniques, 29, 252-256, 2000.

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.

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