Indirect cDNA Labeling for Microarrays

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.

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.

Dye Module
Store at -20° C.

Purification Module

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

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

• 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.

• 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

• 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.

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.

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.
 
Note:    The labeled DNA must be purified as described before scanning, as any unincorporated labeled nucleotides will interfere with the detection of labeled DNA.


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


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


4. 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


5. 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)
   

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