Reverse Transcription System

AMV reverse transcriptase synthesizes single-stranded cDNA from total or poly(A)+ isolated RNA (1). This system provides lot tested reagents to efficiently reverse transcribe RNA in 15 minutes. A polyadenylated 1.2kb transcript is provided as a control template for the cDNA synthesis reaction (see Note 1 ). The system contains sufficient reagents for 100 reactions, processing 1µg of RNA per reaction.

I. Kit Components

• 1500u - AMV Reverse Transcriptase (high concentration)
• 2500u - rRNasin® Ribonuclease Inhibitor
• 50µg - Oligo(dT)₁₅ Primer
• 5µg - Positive Control RNA, 1.2kb
• 320µl - dNTP Mixture, 10mM
• 1.4ml - Reverse Transcription 10X Buffer
• 1.4ml - 25mM MgCl₂
• 25ml - RNase-free water

II. Protocol for Standard Reverse Transcription Reaction (1µg of RNA)

A. Prepare a 20µl reaction by adding the following reagents in order: (Note that this reaction can be scaled up or down depending on the amount of RNA)

Component                            Volume                 Final Concentration  
.                                                           in 20µl               
---------------------------------------------------------------------------------
MgCl2, 25mM                            4µl                  5mM                  
Reverse Transcription 10X Buffer       2µl                  1X = 10mM Tris-HCl   
                                                             (pH 8.8 at 25°C),   
                                                             50mM KCl, and 0.1%  
                                                             Triton® X-100       
10mM dNTP mixture                      2µl                  1mM each dNTP        
rRNasin® Ribonuclease Inhibitor      0.5µl                  1u/µl                
AMV Reverse Transcriptase (H.C.)       15u                  15u/µg               
Oligo(dT)15 Primer                   0.5µg                  0.5µg/µg RNA         
Positive Control RNA or                1µg                  50ng/µl              
  substrate RNA                                                                  
---------------------------------------------------------------------------------
Nuclease-Free Water                   20µl final volume                           

B. Incubate the reaction at 42°C for 15 minutes.

C. To analyze the product on a gel, proceed to Step IV. DO NOT heat the reaction. Heating will cause the RNA/cDNA hybrid to denature, thus creating ambiguous gel results. For other applications the sample should be heated at 99°C for 5 minutes followed by a 5-minute incubation at 0-5°C. This will inactivate the reverse transcription and prevent it from binding to the cDNA.

III. Dilution of the Reaction

A. The first strand cDNA reaction may be diluted in the following manner:

Key Component                        Volume                 Final Concentration  
                                                            in 100µl             
---------------------------------------------------------------------------------
first stand cDNA reaction            20µl                   <10ng/µl             
cDNA reaction dNTPs                                         200µM                
MgCl2, 25mM                           4µl                   1.5mM                
Reverse Transcription 10X Buffer      8µl                   1X = 10mM Tris-HCl   
                                                            (pH 8.8 at 25°C)     
                                                            50mM KCl, and 0.1%   
                                                            Triton X-100         
Nuclease-Free Water                65.5µl                                      
---------------------------------------------------------------------------------
diluted sample volume              97.5µl                                      

IV. Gel Analysis of First Strand cDNA Products

A. To examine the conversion of control RNA to cDNA add 15µl of the reaction to 5µl of the sample buffer and load on a 1% agarose gel containing 0.5µg/ml ethidium bromide. Compared to a sample (approximately 250ng) of the control RNA template there should be a band shift from the lower molecular weight RNA to the higher molecular weight RNA/cDNA hybrid (see Figure 1 ).

Alternatively, a denaturing alkaline agarose gel may be used for gel analysis (2).

Notes:

The Positive Control RNA contains the sequence encoding for kanamycin resistance. Heating the control RNA at 65°C for 5 minutes prior to loading on an agarose gel eliminates any secondary structure. If the sample is not heated the control RNA tends to give a banding pattern of two bands rather than one uniform band.

1. The following reagents may be prepared as a stock just prior to setting up the reactions and aliquoted into individual tubes when needed: water, buffer, dNTPs, MgCl₂ , rRNasin® Ribonuclease Inhibitor, and reverse transcriptase. This allows fewer pipetting steps and improves accuracy.
2. The suggested magnesium concentration may be optimized for any given sequence to achieve better yields.
3. Random primers (available separately, Cat.# C1181) at a concentration of 200ng primer/µg RNA may be substituted for the Oligo(dT) primer in the reverse transcription reaction. When using random primers, incubate the reaction at room temperature for 10 minutes and then incubate at 42°C for 15 minutes. This additional incubation allows extension of the primers so that they remain hybridized when the temperature is raised to 42°C.
4. Specific downstream primers (provided by the user) may also be substituted for the Oligo(dT) primer. The concentration of a specific primer should be adjusted according to the type of reverse transcription being performed. For example, when a 24mer primer is hybridized to 1.0µg of control template RNA, 800ng (100pmol) is required. When the identical primer is hybridized to a specific RNA in a total RNA sample, as little as 120ng (15pmol) is required. Specific primers are typically 19-30 bases long.
5. To obtain longer and/or more abundant transcripts the cDNA reaction may be incubated for up to 60 minutes at 42°C.
6. In cDNA synthesis, significantly fewer units of AMV Reverse Transcriptase are needed for cDNA synthesis relative to some other reverse transcriptases such as MMLV reverse transcriptase.

Solutions:

Sample buffer:

   2% - Ficoll
 0.5% - SDS
 50mM - EDTA
 0.2% - orange G

or

  50% - glycerol
 0.5% - SDS
 0.1% - bromphenol blue
100mM - EDTA

Reverse Transcription 10X Buffer

100mM - Tris-HCl, pH 8.8 at 25°C
500mM - KCl
   1% - Triton X-100

Figure 1. Agarose gel analysis of positive control. First strand cDNA reactions were carried out as described in the text utilizing the provided 1.2kb positive control RNA. Lane 1, pGEM® DNA markers; lane 2, 750ng of 1.2kb Positive Control RNA; lane 3, 15µl of first strand cDNA primed with Oligo(dT)₁₅ ; lane 4, 15µl of first strand cDNA primed with random primers.

References:

1. Goodman, H.M. and MacDonald R.J. (1979) Meth. Enzymol. 68, 75.

2. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Copyright 1995 Promega Corp. All Rights Reserved.
pGEM and RNasin are registered trademarks of Promega Corporation.

All prices and specifications contained in this document are subject to change without prior notice.