Introduction

Overview  
 
Enzymatic incorporation of an amine-modified nucleotide during RT and the subsequent chemical coupling of the cDNA with fluorescent succinimidyl esters is a preferred labeling method for many scientists performing expression analysis on DNA microarrays.

Introduction
 
The SuperScript™ Plus Indirect cDNA Labeling System is a highly efficient system for generating fluorescently labeled cDNA for use on microarrays in gene expression studies.  It uses an aminoallyl-modified nucleotide and an aminohexyl-modified nucleotide together with other dNTPs in a cDNA synthesis reaction with SuperScript™ III Reverse Transcriptase.  After a purification step to remove unincorporated nucleotides, the amino-modified cDNA is coupled with a monoreactive, N-hydroxysuccinimide (NHS)-ester fluorescent dye included in the kit - either Alexa Fluor® 555 succinimidyl ester or Alexa Fluor® 647 succinimidyl ester.  A final purification step removes any unreacted dye, and the fluorescently labeled cDNA is ready for hybridization to microarrays.
 
This system uses 5–20 µg of total RNA or 0.4–2 µg of mRNA as starting material.  Catalog nos. L1014-05 and L1014-06 include a Purification Module containing Low-Elution-Volume Spin Cartridges that yield a highly pure, highly concentrated sample.
 
Advantages of the System

  • Optimized reagents and protocol ensure highly robust and reproducible labeling reactions.
  • SuperScript™ III Reverse Transcriptase in the first-strand synthesis reaction ensures high specificity and high yields of cDNA, as well as more full-length cDNA.
  • Use of two amino-modified nucleotides in the cDNA synthesis reaction results in a greater incorporation of fluorescent dye, an even distribution of fluorescent signal, and higher signal intensity with small amounts of starting material.
  • Alexa Fluor® dyes provide higher correlation coefficients, signal intensities, and signal-to-background ratios than other labeling dyes.
  • System includes all major reagents and materials for preparing Alexa Fluor®-labeled cDNA.


Advantages of SuperScript™ III Reverse Transcriptase


SuperScript™ III Reverse Transcriptase is an engineered version of M-MLV RT with reduced RNase H activity and increased thermal stability.  The enzyme can be used to synthesize first-strand cDNA from total RNA or mRNA at temperatures up to 55 ° C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases.
 
The SuperScript™ III RT in this kit is provided at an optimal concentration and used at an optimal temperature for incorporating amino-modified nucleotides in first-strand cDNA synthesis.
 
Experimental Outline

The flow chart below outlines the experimental steps of the system




:
 

Alexa Fluor® 555 and Alexa Fluor® 647 Reactive Dyes

The Alexa Fluor® 555 and Alexa Fluor® 647 dyes included in this kit are compatible with commonly used microarray scanners, and provide greater signal correlation (R2) values than the spectrally similar Cy™3 and Cy™5 dye pair, improving the resolution of two-color microarray gene expression assays.  The exceptionally bright Alexa Fluor® dyes are also insensitive to pH and are highly water-soluble. The table below shows the excitation and emission maxima and color of each dye:
 
Dye                                               Excitation/Emission (nm)                          Color
Alexa Fluor® 555                                    555/565                                 Orange Fluorescent
Alexa Fluor® 647                                    650/670                                 Far-Red Fluorescent
 
Anchored Oligo(dT)20

Anchored oligo(dT)20 primer is a mixture of 12 primers, each consisting of a string of 20 deoxythymidylic acid (dT) residues followed by two additional nucleotides represented by VN, where V is dA, dC, or dG, and N is dA, dC, dG or dT.

The VN “anchor” allows the primer to anneal only at the 5 US end of the poly(A) tail of mRNA, providing more efficient cDNA synthesis for labeling applications.
 
Control Reaction

We recommend performing the labeling procedure using the Control HeLa RNA included in the system to determine the efficiency of the labeling reaction.  The section on First-Strand cDNA Synthesis describes how to set up the control reaction and Assessing Labeling Efficiency has equations for calculating the efficiency of the labeling procedure.

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Materials

Kit Sizes and Modules

All versions of the SuperScript™ Plus Indirect cDNA Labeling System are supplied with a Core Module and a Dye Module. Catalog nos. L1014-05 and L1014-06 also include a Purification Module.
 
Cat no.            Number of Labeling Reactions                         Modules
L1014-04                                30                                       Core and Dye only
L1014-05                                10                                       Core, Dye, and Purification
L1014-06                                30                                       Core, Dye, and Purification
 
Shipping and Storage

The Core Module and Dye Module are shipped on dry ice, and the Purification Module is shipped at room temperature. Upon receipt, store the components of the Core and Dye Modules at -20°C, and store the components of the Purification Module at room temperature.
 
Core Module

Store at -20° C.

 
 
Kit Size
Item
Components/Concentration
10 Rxns30 Rxns
SuperScript III Reverse Transcriptase
400 U/µl in:
20 mM Tris-HCl (pH 7.5)
100 mM NaCl
0.1 mM EDTA
1 mM DTT
0.01% (v/v) NP-40
50% (v/v) glycerol
20 µl60 µl
5X First-Strand Buffer
250 mM Tris-HCl (pH 8.3, room temp)
375 mM KCl
15 mM MgCl2
60 µl200 µl
Dithiothreitol (DTT)
0.1 M DTT in water
250 µl250 µl
dNTP Mix
dATP, dGTP, dCTP, dTTP, one aminoallyl-modified nucleotide, and one aminohexyl-modified nucleotide in DEPC-treated water
15 µl45 µl
2X Coupling Buffer
50 µl300 µl
Anchored Oligo(dT)20 primer
2.5 µg/µl in DEPC-treated water
20 µl60 µl
Random hexamer primers
0.5 µg/µl in DEPC-treated water
10 µl30 µl
DMSO
200 µl750 µl
RNaseOUT
40 U/µl
10 µl30 µl
DEPC-treated Water
2 ml6 ml
Control HeLa RNA
1 µg/µl
20 µl20 µl


Dye Module
Store at -20° C.

Item
Components/Concentration
Kit Size
  10 Rxns30 Rxns
Alexa Fluor® 555 Reactive Dye Pack
60 µg dried-down dye per vial
5 vials3 x 5 vials
Alexa Fluor® 647 Reactive Dye Pack
60 µg dried-down dye per vial
5 vials3 x 5 vials


Purification Module

Store at room temperature. This module is included with Catalog Numbers L1014-05 and L1014-06.

 
Kit Size
Component
10 Rxns30 Rxns
Low-Elution Volume Spin Cartridges (with collection tubes)
2 x 11 columns6 x 11 columns
Binding Buffer (must be combined with 100% isopropanol to create final buffer; see Preparing the Buffers)
2 x 5.5 ml2 x 18 ml
Wash Buffer (must be combined with 100% ethanol to create final buffer; see Preparing the Buffers)
2 x 2 ml2 x 5 ml
Amber collection tubes
2 x 11 tubes6 x 11 tubes



Materials Supplied by the User

In addition to the kit components, you should have the following items on hand before using the SuperScript™ Indirect cDNA Labeling System.

  • Vortex mixer
  • Microcentrifuge
  • Aerosol resistant pipette tips
  • Water baths or incubator
  • 1 N NaOH
  • 1 N HCl
  • Sterile microcentrifuge tubes
  • 100% Isopropanol
  • 100% Ethanol
  • 75% Ethanol
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Ordering Information

Sku Name Size Price Qty
L101404 SuperScript® Plus Indirect cDNA Labeling Module (no purification) with Alexa Fluor® Dyes 30 reactions USD 890.00
L101406 SuperScript® Plus Indirect cDNA Labeling System with Alexa Fluor® Dyes 30 reactions USD 919.80

Isolating RNA

High-quality, intact RNA is essential for full-length, high-quality cDNA synthesis.  In this step, you isolate total RNA or mRNA using a method of choice.
 
The quality of the RNA is critical for successful labeling and hybridization.  The presence of contaminants in the RNA may significantly increase background fluorescence in your microarrays.  Carefully follow the recommendations below to prevent RNase contamination.
 
General Handling of RNA
 
When working with RNA:

  • Use disposable, individually wrapped, sterile plasticware.
  • Use aerosol resistant pipette tips for all procedures.
  • Use only sterile, new pipette tips and microcentrifuge tubes.
  • Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin.
  • Use proper microbiological aseptic technique when working with RNA.
  • Dedicate a separate set of pipettes, buffers, and enzymes for RNA work.
  • Microcentrifuge tubes can be taken from an unopened box, autoclaved, and used for all RNA work. RNase-free microcentrifuge tubes are available from several suppliers.  If it is necessary to decontaminate untreated tubes, soak the tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC-treated), rinse the tubes with sterile distilled water, and autoclave the tubes.

You can use RNase Away™ Reagent, a non-toxic solution available from Invitrogen (see page vii), to remove RNase contamination from surfaces. For further information on controlling RNase contamination, see Ausubel, et al., 1994, and Sambrook, et al., 1989. (Ausubel et al., 1994; Sambrook et al., 1989)

Isolating RNA

This system is optimized for use with 10–40 µg total RNA or 0.4–2 µg of mRNA.  Lower amounts of starting material may be used, but may result in lower hybridization signals.
 
To isolate total RNA, we recommend the PureLink™ Micro­-to-Midi Total RNA Purification System, TRIzol® Reagent, or (for high-throughput applications) the PureLink™ 96 RNA Purification System.  To isolate mRNA, we recommend the FastTrack® 2.0 mRNA Isolation Kits or the FastTrack® MAG mRNA Isolation Kits.  After you have isolated the RNA, check the quality of your RNA preparation. as described on the following age.
 
Checking the RNA Quality

To check RNA quality, analyze 500 ng of RNA by agarose/ethidium bromide gel electrophoresis.  You can use a regular 1% agarose gel or a denaturing agarose gel (Ausubel et al., 1994). For total human RNA using a regular agarose gel, mRNA will appear as a smear from 0.5 to 9 kb, and 28S and 18S rRNA will appear as bands at 4.5 kb and 1.9 kb, respectively.  The 28S band should be twice the intensity of the 18S band.  If you are using a denaturing gel, the rRNA bands should be very clear and sharp.
 
If you do not load enough RNA, the 28S band may appear to be diffuse.  A smear of RNA or a lower intensity 28S band with an accumulation of low molecular weight RNA on the gel are indications that the RNA may be degraded, which will decrease the labeling efficiency.  If you do not detect any RNA, you will need to repeat RNA isolation. Refer to the Troubleshooting section.

Storing RNA

After preparing the RNA, we recommend that you proceed directly to First-Strand cDNA Synthesis.  Otherwise, store the RNA at –80 °C.
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First-Strand cDNA Synthesis

After you have isolated RNA and checked the quality of your RNA preparation, you are ready to synthesize cDNA.
 
Before Starting

The following materials are supplied by the user:

  • 5–20 µg total RNA or 0.4–2 µg mRNA
  • 1 N NaOH
  • 1 N HCl
  • Water baths, heating block, or incubator set at 46°C and 70°C
  • Ice
  • 1.5-ml RNase-free microcentrifuge tubes

The following materials are supplied in the kit:

  • Anchored Oligo(dT)20 primer
  • Random hexamers (for mRNA starting material only)
  • dNTP mix, including amino-modified nucleotides
  • 5X First-Strand buffer
  • 0.1 M DTT
  • RNaseOUT™
  • SuperScript™ III RT
  • DEPC-treated water
  • 10 µg of Control HeLa RNA per reaction; optional
  • RNaseOUT™ Recombinant RNase Inhibitor has been included in the system to safeguard against degradation of target RNA due to ribonuclease contamination of the RNA preparation.

First-Strand cDNA Synthesis

The following procedure is designed to convert 5–20 µg of total RNA or 0.4–2 µg of mRNA into first-strand cDNA.
 
Note:   If you are setting up a control reaction (recommended for first-time users), use 10 µl of the Control HeLa RNA supplied in the kit (1 µg/µl).
 
  1.     Mix and briefly centrifuge each component before use.
 
  2.     Prepare each reaction as follows in a 1.5-ml RNase-free tube:

Component Volume
5–20 µg total RNA or 0.4–2 µg mRNAX µl
Anchored Oligo(dT)20 Primer (2.5 µg/µl)2 µl
Random hexamers (only if using mRNA) 1 µl *
DEPC-treated water to 18 µl

*For mRNA, use both anchored oligo(dT)20 and random hexamers. For total RNA, use only 2 µl of anchored oligo(dT)20.
 
  3.    Incubate tube at 70 °C for 10 minutes, and then place on ice for at least 1 minute.
 
  4.    Add the following to the tube on ice:

Component Volume
5X First-Strand buffer
  6 µl
0.1 M DTT1.5 µl
dNTP mix (including amino-modified nucleotides) 1.5 µl
RNaseOUT™ (40 U/µl)    1 µl
SuperScript™ III RT (400 U/µl)
   2 µl
Final Volume 
30 µl

  5.     Mix gently and collect the contents of each tube by brief centrifugation. Incubate tube at 46°C for 2­–3 hours.
 
Note:    A 3-hour incubation results in 20–30% higher cDNA yield than a 2-hour incubation.
 
After incubation, proceed directly to Alkaline Hydrolysis and Neutralization, below.
 
Alkaline Hydrolysis and Neutralization 

After cDNA synthesis, immediately perform the following hydrolysis reaction to degrade the original RNA:
 
  1.      Add 15 µl of 1 N NaOH to each reaction tube from Step 5, above. Mix thoroughly.

  2.      Incubate tube at 70°C for 10 minutes.

  3.      Add 15 µl of 1 N HCl to neutralize the pH and mix gently.
 
Proceed to Purifying the First-Strand cDNA
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Purifying the Labeled cDNA

Catalog nos. L1015-05 and L1015-06 include a Purification Module developed for use with the system.  Follow the procedure below to purify your labeled cDNA using this module.
 
Catalog no. L1015-04 does not include a Purification Module.  Use your preferred method of cDNA purification instead of the following procedure, and then continue to hybridization.
 
The PureLink™ PCR Purification System (K3100-01 and K3100-02) has been tested with this kit, and is recommended if you are using catalog no. L1015-04.
 
Before Starting

The following items are supplied by the user:

  • Microcentrifuge


 The following items are supplied in the Purification Module:

  • DEPC-treated water
  • Low-Elution Volume Spin Cartridges pre-inserted into collection tubes
  • Amber collection tubes
  • Binding Buffer (prepared with isopropanol as describedon page vi)
  • Wash Buffer (prepared with ethanol)


Purification Procedure

Use the following procedure to purify the cDNA using the components of the Purification Module (Catalog nos. L1015-05 and L1015-06).
 

  1. Add 700 µl of Binding Buffer (prepared with isopropanol) to the reaction tube containing the labeled cDNA (from the Hydrolysis and Neutralization procedure).
  2. Each Low-Elution Volume Spin Cartridge is preinserted into a collection tube.  For multiple reactions, clearly label each collection tube, and then load the cDNA/Binding Buffer solution directly onto the Spin Cartridge.
  3. Centrifuge at 3,300 x g in a microcentrifuge for 1 minute.  Remove the collection tube and discard the flow-through.
  4. Place the Spin Cartridge in the same collection tube and add 600 µl of Wash Buffer (prepared with ethanol to the column.
  5. Centrifuge at maximum speed for 30 seconds.  Remove the collection tube and discard the flow-through.
  6. Place the Spin Cartridge in the same collection tube and centrifuge at maximum speed for 30 seconds to remove any residual Wash Buffer.  Remove the collection tube and discard.
  7. Place the Spin Cartridge onto a new amber collection tube (supplied in the kit).
  8. Add 20 µl of DEPC-treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute.
  9. Centrifuge at maximum speed for 1 minute to collect the purified labeled cDNA.  The eluate contains your purified labeled cDNA.

 
The sample can stored at –20° C for up to one week prior to hybridization.  Avoid freeze/thawing.  To determine the efficiency of the labeling reaction, proceed to Assessing Labeling Efficiency.
 
Because of the high purity of the cDNA from the Low-Elution Volume Spin Cartridges included with catalog nos. L1015-05 and L1015-06, the yield and picomole dye incorporation calculations will be more accurate than with other purification methods.

For example, the 1.2% E-Gel below shows purification results from an indirect labeling method.  Lanes 1 and 2 contain Alexa Fluor® 555-labeled cDNA purified using the Low-Elution Volume Spin Cartridges, and Lanes 3 and 4 contain Alexa Fluor® 555-labeled cDNA purified using columns from another manufacturer.  The labeled cDNA appears as smear from 500–5,000 bp.

The large band at the bottom of Lanes 3 and 4 is unincorporated dye that was not removed by the other manufacturer’s purification column.  Such material would be included in the picomole dye incorporation calculations, resulting in an incorporation level that is higher than theoretically possible.

For this reason, we strongly recommend using the purification columns provided with catalog nos. L1015-05 and L1015-06.
 

 

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Support Protocol 1: Assessing Labeling Efficiency

You can use UV/visible spectroscopy scanning to measure the amount of labeled cDNA and dye incorporation.  The expected amounts using the Control HeLa RNA provided in the kit are shown below.
 
Calculating the Results

To calculate the amount of labeled cDNA using a UV/visible spectrophotometer:
 
  1. Transfer a volume of purified, labeled cDNA from step 9, to a clean cuvette.  Use an appropriate volume for your spectrophotometer.  Add DEPC-treated water to the cDNA if you need to increase the volume of the eluate for your spectrophotometer.
  2.  
    Note:    The labeled DNA must be purified as described before scanning, as any unincorporated labeled nucleotides will interfere with the detection of labeled DNA.

  3. Blank the spectrophotometer using DEPC-treated water, and then scan the sample at 240–800 nm.  Wash each cuvette thoroughly between samples.

  4. Calculate the yield of cDNA using the following formula:
     
    cDNA (ng) = (A260–A320) x 37 ng/µl x volume in µl

  5. Calculate the amount of fluorescent dye using the following formulas:
     
    Alexa Fluor® 555 (pmole) = (A555–A650)/0.15 x volume in µl
    Alexa Fluor® 647 (pmole) = (A650–A750)/0.24 x volume in µl

  6. Calculate the base-to-dye ratio using the following formulas:
     
    Base/dye ratio for Alexa Fluor® 555 =
     
    {(A260 – A320) – [(A555 – A650) x 0.04]} x 150,000/(A555 – A650) x 8,919
     
    Base/dye ratio for Alexa Fluor® 647 =
     
    {(A260 – A320) – [(A650 – A750) x 0]} x 239,000/(A650 – A750) x 8,919
     
    The number of dye molecules per 100 bases is calculated using the formula:

    100/(base/dye ratio)
 
Expected Amounts Using Control DNA

If you prepare a control reaction using 10 µg of Control HeLa RNA as starting material, the following amounts are expected:
 
             Labeled                        Incorporated Labeled          Dyes Molecules/
              cDNA                                  Nucleotides                           100 Bases

            > 400 ng                               > 30 pmole                                 > 1.0
 
If you do not obtain these amounts, see Troubleshooting.
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Troubleshooting

Problem
Cause
Solution
28S and 18S bands are not observed after isolation of total RNA and agarose gel electrophoresis
Too little RNA loaded on the gel
Be sure to load at least 250 ng of RNA for analysis.
 
RNA is degraded due to RNase activity
Follow the guidelines to avoid RNase contamination.
Use a fresh sample for RNA isolation.
28S band is diminished or low molecular weight RNA appears in the gel
RNA is degraded
Follow the guidelines to avoid RNase contamination.
Use a fresh sample for RNA isolation.
Yield of cDNA is low
Temperature too high during cDNA synthesis
Perform the cDNA synthesis at 46°C.
 
Incorrect reaction conditions used
Verify that all reaction components are included in the reaction and use reagents provided in the system.
Verify the reaction conditions using the Control HeLa RNA provided in the kit.
 
Concentration of template RNA is too low
Increase the concentration of template RNA. Use at least 10 µg of total RNA or 0.4 µg of mRNA.
 
Poor quality RNA used or RNA is degraded
Check the quality of your RNA preparation. If RNA is degraded, use fresh RNA.
 
RNase contamination
Use the RNaseOUT included in the kit to prevent RNA degradation.
 
RT inhibitors are present in your RNA sample
Inhibitors of RT include SDS, EDTA, guanidinium chloride, formamide, sodium phosphate and spermidine (Gerard, 1994). Test for the presence of inhibitors by mixing 1 µg of Control HeLa RNA with 25 µg total RNA or 1 µg mRNA and compare the yields of first-strand synthesis.
 
Improper storage of SuperScript III RT
Store the enzyme at -20° C.
 
Reagents were not properly mixed before use.
Repeat the procedure, being careful to briefly vortex and centrifuge each reagent before use.
 
cDNA has been lost in the purification step
Measure the amount of cDNA produced by the Control RNA before and after purification. Follow the purification procedure without modifications.
Amount of incorporated labeled nucleotides in the control reaction is low and/or fluorescence of labeled cDNA is low
Reaction tubes have been exposed to light
Avoid direct exposure of the labeling reaction to light. Use the amber tube provided in the kit for collection of the final product.
 
Inefficient labeling due to improper purification
Follow all purification steps carefully and without modification.
 
Starting amount of RNA is too low
Increase the amount of starting RNA

 

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References

  1. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).

  2. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. Z. (1979). Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonucleases. Biochem. 18, 5294-5299.

  3. Chomczynski, P., and Sacchi, N. (1987). Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem. 162, 156-159.

  4. De Risi, J., Penland, L., Brown, P.O., Bittner, M.L., Meltzer, P.S., Ray, M., Chen, Y., Su, Y.A., Trent, J.M. (1996) Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nature Genet. 14, 457–460.

  5. Eisen M.B., Brown P.O. (1999) DNA arrays for analysis of gene expression. Methods Enzymol 303,179–205.

  6. Gerard, G. F. (1994). Inhibition of SuperScript II Reverse Transcriptase by Common Laboratory Chemicals. Focus® 16, 102-103.

  7. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). 
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LT065