Introduction

Overview

The BioModule™ Microarray Units provide qualified reagents and validated protocols for fluorescent labeling of cDNA synthesized from purified total RNA, and subsequent hybridization of the labeled cDNA to DNA microarrays for gene expression profiling (De Risi et al., 1996; Eisen & Brown, 1999).
 
The Indirect cDNA Labeling Unit includes the SuperScript™ Plus Indirect cDNA Labeling System for generating labeled cDNA using aminoallyl- and aminohexyl-modified nucleotides and either Alexa Fluor® 555 succinimidyl ester or Alexa Fluor® 647 succinimidyl ester.
 
The Direct cDNA Labeling Unit includes the SuperScript™ Plus Direct cDNA Labeling System for generating labeled cDNA using nucleotide mixes that contain Alexa Fluor® 555-aha-dUTP or Alexa Fluor® 647-aha-dUTP.
 
Both units include TRIzol® Reagent and the PureLink™ Micro-to-Midi Total RNA Purification Kit for high-quality, rapid purification of total RNA, and SuperScript™ III Reverse Transcriptase (RT) for high-temperature synthesis of first-strand cDNA from 5–20 µg of total RNA. Both units also include optimized microarray hybridization buffers that are compatible for use with Corning® GAPS, GAPS II, and UltraGAPS™ slides.
 
Summary of Components

Each BioModule™ Microarray Unit includes:

  • TRIzol® Reagent and the PureLink™ Micro-to-Midi Total RNA Purification System, for isolating total RNA from mammalian cells, fresh and frozen tissues, whole blood, and liquid samples.
  • Deoxyribonuclease I (DNase I), Amplification Grade, for eliminating DNA during RNA purification procedures
  • SuperScript™ III Reverse Transcriptase for high-temperature cDNA synthesis, ensuring high specificity and high yields of cDNA as well as more full-length cDNA
  • 5X First-Strand Buffer and anchored oligo(dT)20 primer for cDNA synthesis
  • RNaseOUT™ Recombinant Ribonuclease Inhibitor, to safeguard against the degradation of target RNA due to ribonuclease contamination.
  • Alexa Fluor®-labeled nucleotides or amine-modified nucleotides and Alexa Fluor® dyes, for labeling cDNA
  • Spin cartridges and buffers for purifying the labeled cDNA
  • Optimized array hybridization and wash buffers. For more information about each reagent


Labeling and Hybridization Workflow Overview

To use the BioModule™ Microarray Unit, you will:

  • Purify total RNA from lysate using TRIzol® Reagent and the PureLink™ Micro-to-Midi Total RNA Purification System, and treat with DNase I
  • Determine RNA quality and yield
  • Synthesize and label cDNA using indirect or direct labeling
  • Purify the labeled cDNA
  • Hybridize the labeled cDNA to microarrays
  • Analyze the results



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Materials

Types of Products

Product
                 Catalog no.
BioModule Microarray Unit
with Indirect cDNA Labeling
with Direct cDNA Labeliing
WFGE03
WFGE04


Number of Boxes

The number of boxes provided with each unit is listed below

Components
WFGE03
WFGE04
SuperScript Plus Indirect cDNA Labeling System
Core Module
Dye Module
Purification Module

1        
1        
1
SuperScript Plus Direct cDNA Labeling System
Core Module
Dye Module
Purification Module

1
1
1
DNase I, amplification grade
22
PureLink Micro-to-Midi Total RNA Purification Kit
11
TRIzol® Reagent
11
UltraPure Formamide
11
BioModule Microarray Hybridization Solutions
11


Shipping and Storage

The shipping conditions for each component are listed below. Upon receipt, store the components as described.


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Component
Shipping
               Storage
TRIzol® Reagent
Room temp.
2–8ºC
PureLink Micro-to-Midi Total RNA Purification System
Room temp.
Room temp.
DNase I, amplification grade
Dry ice
–20ºC in a non-frost free freezer
SuperScript Plus Indirect cDNA Labeling System
Core Module
Dye Module
Purification Module

Dry ice
Dry ice
Room temp.

–20ºC
–20ºC
Room temp.
SuperScript Plus Direct cDNA Labeling System
Core Module
Dye Module
Purification Module

Dry ice
Dry ice
Room temp.

–20ºC
–20ºC
Room temp.
BioModule Microarray Hybridization Solutions
Room temp.
2–8ºC
UltraPure Formamide
Dry ice
–20ºC


TRIzol® Reagent

This unit includes 100 ml of TRIzol® Reagent, which is sufficient for ~100 isolations.

PureLink™ Micro-to-Midi Total RNA Purification

The components provided with the PureLink™ Micro-to-Midi Total RNA Purification System are described below. Sufficient reagents are provided to perform 50 purifications.

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ItemAmount
RNA Lysis Solution125 ml
Wash Buffer I50 ml
Wash Buffer II15 ml
RNase-Free Water15 ml
TRNA Spin Cartridges, with collection tubes50
RNA Wash Tubes50
RNA Recovery Tubes50


DNase I, Amplification Grade
 
Two boxes of DNase I, Amplification Grade, are provided with each unit. Components are listed below. Sufficient reagents are provided for 40 on-column DNase I treatments.
 
Item                                                                         Amount
DNase I, Amp Grade                                             2 × 100 µl
10X DNase I Reaction Buffer                               2 × 100 µl
25 mM EDTA (pH 8.0)                                           2 × 100 µl

10X DNase I Reaction Buffer
10X DNase I Reaction Buffer contains 200 mM Tris-HCl (pH 8.4), 20 mM MgCl2, and 500 mM KCl.
 
Microarray Hybridization Solutions

The BioModule™ Microarray Hybridization Solutions are described below. Formulations are proprietary. Sufficient reagents are provided to perform 30 hybridizations. 

Item
Components/Concentration
          Amount
Pre-Hybridization
Solution
Combine with Buffer WB and formamide
129.5 ml
Hybridization Solution
Combine with Buffer WB and formamide
720 µl
Buffer WA
2 × 240 ml
Buffer WB
85 ml


UltraPure™ Formamide

This unit includes 500 g of UltraPure™ Formamide in liquid form, which must be added to the Hybridization Solution before use.
 
SuperScript™ Plus Indirect cDNA Labeling System

The SuperScript™ Plus Indirect cDNA Labeling System is included with the BioModule™ Microarray Unit with Indirect cDNA labeling (Catalog no. WFGE03). Sufficient reagents are provided to perform 30 labeling reactions.


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Item
Components/Concentration
         30 Rxns
Core Module
SuperScript III Reverse Transcriptase
400 U/µl
60 µl
5X First-Strand Buffer
250 mM Tris-HCl (pH 8.3, room temp), 375 mM KCl, 15 mM MgCl2
200 µl
Dithiothreitol (DTT)
0.1 M DTT in water
250 µl
dNTP Mix
dATP, dGTP, dCTP, dTTP, one aminoallyl-modified nucleotide, and one aminohexyl-modified nucleotide
45 µl
2X Coupling Buffer
300 µl
Anchored Oligo(dT)20 primer
2.5 µg/µl in DEPC-treated water
60 µl
Random hexamer primers
0.5 µg/µl in DEPC-treated water
30 µl
DMSO
750 µl
RNaseOUT
40 U/µl
30 µl
DEPC-treated Water
6 ml
Control HeLa RNA
1 µg/µl
20 µl
Dye Module
Alexa Fluor® 555 Reactive Dye Pack
60 µg dried-down dye per vial
3 × 5 vials
Alexa Fluor® 647 Reactive Dye Pack
60 µg dried-down dye per vial
3 × 5 vials
Purification Module
Low-Elution Volume Spin Cartridges
 Pre-inserted into collection tubes
6 × 11 columns
Binding Buffer
Must be combined with 100% isopropanol to create final buffer; see page 14
2 × 18 ml
Wash Buffer
Must be combined with 100% ethanol to create final buffer; see page 14
2 × 5 ml
Amber collection tubes
6 × 11 tubes

 

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Total RNA Purification

Introduction

This section provides guidelines and protocols for total RNA purification from animal cells or tissues using TRIzol® Reagent, the PureLink™ Micro-to-Midi Total RNA Purification System, and DNase I, Amplification Grade.
 
The protocols in this section use TRIzol® Reagent, the PureLink™ Micro-to-Midi Total RNA Purification System, and DNase I, Amplification Grade. Have all components from these boxes ready before proceeding.
 
Guidelines for Handling RNA

Follow the guidelines below to prevent RNase contamination and to maximize the RNA yield: 

  • Use disposable, individually wrapped, sterile plastic ware.
  • Use only sterile, disposable RNAse-free pipette tips and microcentrifuge tubes.  
  • Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. Change gloves frequently, particularly as the purification protocol progresses from crude extracts to more purified material.
  • Always use proper microbiological aseptic techniques when working with RNA.
  • Use RNase AWAY® Reagent to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.  

Guidelines for Sample Collection

Use the following guidelines for collecting your samples to minimize RNA degradation prior to RNA purification and to maximize the RNA yield:
 
  • Always wear disposable gloves while handling samples and reagents to prevent RNase contamination.
  • Work quickly during sample harvesting, use RNase-free dissection tools and containers (scalpels, dishes, tubes etc.) and work on RNAse-free work surfaces (use RNase AWAY® Reagent).
  • To purify total RNA from fresh samples, keep fresh cell and tissue samples on ice immediately after harvesting; quickly proceed to adding RNA Lysis solution, sample lysis and homogenizaton.
  • To purify total RNA from frozen samples, freeze samples immediately after harvesting in liquid nitrogen or on dry ice. Keep frozen samples at -80° or in liquid nitrogen until proceeding to sample lysis and homogenization.  
  • Whole blood: Process freshly drawn blood immediately, and keep at room temperature until processing.

 
TRIzol® Reagent contains phenol and guanidine isothiocyanate. Toxic in contact with skin and if swallowed. Causes burns. Always wear a laboratory coat, disposable gloves, and eye protection when handling solutions containing this chemical. After contact with skin, wash immediately with plenty of detergent and water. Use in a chemical fume hood. Avoid breathing vapor. Do not add bleach or acidic solutions directly to solutions containing guanidine isothiocyanate or sample preparation waste. Guanidine isothiocyanate forms reactive compounds and toxic gases when mixed with bleach or acids.

Solutions containing ethanol are considered flammable. Use appropriate precautions when using these solutions. 
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Use polypropylene tubes when working with TRIzol® Reagent. Do not use polystyrene tubes. 
 
Frozen tissue must remain frozen at –80°C prior to lysis. Cool tubes in dry ice before placing frozen tissue in them. Thawing of frozen tissue prior to lysis may result in RNA degradation and loss of RNA yield.
 
Additional Materials Needed:

You will need the following additional items not provided with this unit:

  • DEPC-treated water
  • Chloroform (molecular biology grade)
  • 70% ethanol (in RNase-free water)
  • 15-ml  and 1.5-ml RNase-free polypropylene microcentrifuge tubes  (Polypropylene is required for use with TRIzol® Reagent; do not use polystyrene tubes. Round-bottom 15-ml tubes are required for homogenization of tissues using a power homogenizer.)
  • Microcentrifuge capable of centrifuging 12,000 × g
  • Vortex mixer
  • For tissue samples: Power homogenizer with a rotating tip that fits a 15-ml tube (e.g., Ultra Turrax® or Polytron® Homogenizer )
  • 1.5 ml RNase-free tubes 
  • RNase-free pipette tips

Preparing Wash Buffer II with Ethanol

Before using Wash Buffer II from the PureLink™ Micro-to-Midi Total RNA Purification System for the first time, add 60 ml of 96–100% ethanol directly to the bottle. Check the box on the Wash Buffer II label to indicate that ethanol was added.

Lysis and Homogenization of Cells

Follow the steps below to prepare lysates from up to 1 × 106 cells:
 
  1. Adherent cells: Lift cells from the plate by adding TE and resuspend in culture media. Count the cells, and transfer ≤1 × 106 cells to a 15-ml polypropylene centrifuge tube.  Suspension cells: Transfer ≤1 × 106 cells to a 15-ml polypropylene centrifuge tube.

  2. Centrifuge at 2,000 × g for 5 minutes at room temperature to pellet the cells.

  3. Carefully pipet the supernatant from the tube, leaving no more than 30 µl of supernatant. Take care not to disturb the cell pellet.

  4. Immediately add 1 ml of TRIzol® to the tube. Vortex until the cell pellet is completely lysed and no visible particulate matter remains. If necessary, pipet the pellet up and down to disperse.

  5. Transfer the lysate to a new, RNase-free 1.5-ml polypropylene microcentrifuge tube.

Proceed to Total RNA Purification Procedure (below).
 
Lysis and Homogenization of Tissues

Follow the steps below to prepare lysates from animal tissues. Use a power homogenizer with a rotating tip that fits a 15-ml round-bottom tube.
 
Note:    The volume of tissue should not exceed 10% of the volume of TRIzol® Reagent used for homogenization. Keep frozen tissues on dry ice until just prior to homogenization.
 
  1. Add 1 ml of TRIzol® Reagent for every 50–100 mg of tissue to a 15-ml round-bottom polypropylene microcentrifuge tube. Immediately add fresh or frozen tissue to the tube and quickly homogenize the sample using a power homogenizer at a medium setting. Homogenize using short bursts for ~2 minutes. Avoid foaming by keeping the rotating tip submerged in solution while holding the tip against the tube wall. Keep homogenate on ice.

  2. Transfer 1-ml aliquots of homogenate to separate 1.5-ml polypropylene microcentrifuge tubes on ice.

  3. Centrifuge tubes at 2,000 × g for 5 minutes at 5°C to pellet cellular debris.

  4. Carefully transfer the supernatant (~800 µl) containing the RNA from each tube to a new, RNase-free 1.5-ml polypropylene tube.

  5. Add enough TRIzol® (~200 µl) to the tube to bring the total volume to 1 ml. Proceed to Total RNA Purification Procedure (below).
 
 
Total RNA Purification Procedure

Follow the steps below to purify your total RNA sample:
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  1. Add 0.2 ml of chloroform to each 1.5-ml tube from Step 5 of the lysis and homogenization protocols above. Mix well by vortexing.

  2. Incubate at room temperature for 2 minutes to allow for phase separation.

  3. Centrifuge the tube at 12,000 × g for 15 minutes at 5°C. After centrifugation, a clear aqueous upper phase and pink organic lower phase should be visible. Some white flocculent material may also be visible in the interface between the phases.

  4. Using a pipette, very carefully remove the upper phase (up to 0.5 ml) without disturbing the lower phase or interface. Take care not to remove any flocculent material with the upper phase. (It is better to remove less of the upper phase if there is a danger of disturbing the lower phase or interface.) Transfer the upper phase to a new RNase-free 1.5-ml tube.

  5. To the volume of upper-phase solution in the new tube, add an equal volume of 70% ethanol by adding ½ volumes sequentially, and mixing after each addition. Mix carefully to avoid precipitation of RNA due to local concentrations of ethanol.

  6. Transfer up to 700 µl of the sample to a PureLink™ RNA Spin Cartridge pre-inserted in a collection tube. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.

  7. Repeat Step 6 until the entire sample has been processed.

  8. Add 350 µl of Wash Buffer I to the spin cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.

  9. In a separate RNase-free tube, prepare a DNase I solution by adding:
  10.  
    DNase I, Amplification Grade                                                                              5 µl
    10X DNase I Reaction Buffer                                                                              5 µl
    DEPC-treated water                                                                                          40 µl

  11. Add the entire volume of DNase I solution directly onto the spin cartridge. Incubate at room temperature for 15 minutes.

  12. Add 350 µl of Wash Buffer I to the spin cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the collection tube.

  13. Place the spin cartridge into a clean RNA Wash Tube, provided in the kit.

  14. Add 500 µl Wash Buffer II with ethanol to the spin cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.

  15. Repeat Step 13 once.

  16. Centrifuge the spin cartridge at 12,000 × g for 1 minute to dry the membrane with attached RNA. Discard the collection tube, and insert the cartridge into an RNA Recovery Tube supplied with the kit.

  17. To elute the RNA, add 30 µl of RNase-free water to the center of the spin cartridge membrane, and incubate at room temperature for 1 minute.

  18. Centrifuge the spin cartridge for 2 minutes at ≥12,000 × g  to collect the eluate.

  19. Remove the recovery tube and add 10 µl of DEPC-treated water to the eluate. Add this solution back onto the spin cartridge membrane. Re-insert the cartridge in the recovery tube, and incubate at room temperature for 1 minute.

  20. Centrifuge the spin cartridge for 2 minutes at ≥12,000 × g to collect the eluate containing the total RNA.
    Determine the quantity and quality of the RNA or store the purified total RNA at –80°C
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Determining RNA Yield and Quality

Introduction

After you have purified the total RNA, determine the quantity and quality as described in this section.
 
Determining RNA Yield

Total RNA is easily quantitated using the Quant-iT™ RiboGreen® RNA Assay Kit or UV absorbance at 260 nm.
 
Quant-iT™ RiboGreen® RNA Assay Kit

The kit contains a state-of-the-art quantitation reagent and pre-diluted standards for a standard curve. The assay is performed in a microtiter plate format and is designed to be read using a standard fluorescent microplate reader.
 
UV Absorbance

To determine the quantity by UV absorbance:
 
  1. Dilute an aliquot of the total RNA sample in 10 mM Tris-HCl, pH 7.5. Mix well. Transfer to a cuvette (1-cm path length). Note: The RNA must be in a neutral pH buffer to accurately measure the UV absorbance.

  2. Determine the A260 of the solution using a spectrophotometer blanked against 10 mM Tris-HCl, pH 7.5. Calculate the amount of total RNA using the following formula:
 
Total RNA (µg) = A260 × [40 µg/(1 A260 × 1 ml)] × dilution factor × total sample volume (ml)
 
Example: 

Total RNA was eluted in water in a total volume of 150 µl. A 40-µl aliquot of the eluate was diluted to 500 µl in 10 mM Tris-HCl, pH 7.5. An A260 of 0.188 was
obtained. The amount of RNA in the sample is determined as shown below:
 
Total RNA (µg) = 0.188 × [40 µg/(1 A260 × 1 ml)] × 12.5 × 0.15 = 14.1 µg

Analyzing RNA Quality

Bioanalyzer:
    RNA quality can be analyzed with an Agilent Bioanalyzer, using a 200 ng/µl dilution of total RNA. Follow the instrument manufacturers instructions.
 
Gel Electrophoresis:  Quality may also be analyzed by agarose/ethidium bromide gel electrophoresis. We recommend using 500 ng of RNA loaded onto a 1.2% E-Gel™ agarose gel. For total human RNA using an 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.
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Indirect cDNA Labeling

Introduction

This section provides protocols for using the SuperScript™ Plus Indirect cDNA Labeling System, included with BioModule™ catalog no. WFGE03.

Using this system, you synthesize first-strand cDNA from total RNA using aminoallyl-modified nucleotides and aminohexyl-modified nucleotides together with other dNTPs in a 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—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.
 
Additional Materials Needed

In addition to the SuperScript™ Indirect cDNA Labeling System components, you will need the following items:

  • Vortex mixer
  • Microcentrifuge
  • Water baths, heating blocks, or incubator
  • Aerosol resistant RNase-free pipette tips
  • 1.5-ml RNase-free microcentrifuge tubes
  • 1 N NaOH
  • 1 N HCl
  • 100% Isopropanol
  • 100% Ethanol
  • 75% Ethanol

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.
 
Amount of RNA

This system is optimized for use with 5–20 µg total RNA. Lower amounts of starting material may be used, but may result in lower hybridization signals.
 
Alexa Fluor® 555 and Alexa Fluor® 647 Reactive Dyes

The Alexa Fluor® 555 and Alexa Fluor® 647 dyes are compatible with commonly used microarray scanners. The table below shows the excitation and emission maxima and color of each dye:
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Dye Excitation/Emission (nm) Color
Alexa Fluor® 555 555/565Orange Fluorescent
Alexa Fluor® 647 650/670 Far-Red Fluorescent


5X First-Strand Buffer


The 5X First-Strand Buffer includes 250 mM Tris-HCl (pH 8.3 at room temperature), 375 mM KCl, and 15 mM MgCl2.

Preparing Binding Buffer with Isopropanol

The Binding Buffer supplied with the Purification Module must be mixed with 100% isopropanol prior to use. Add the amount of isopropanol indicated below directly to the bottle of Binding Buffer to create the final buffer. Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the isopropanol.

  10-rxn kit 30-rxn kit
Binding Buffer 5.5 ml (entire bottle)18.0 ml (entire bottle)
100% Isopropanol 2.0 ml   6.5 ml
Final Volume 7.5 ml 24.5 ml

Store the Binding Buffer prepared with isopropanol at room temperature.
 
Preparing Wash Buffer with Ethanol

The Wash Buffer supplied with the Purification Module must be mixed with 100% ethanol prior to use. Add the amount of ethanol indicated below directly to the bottle of Wash Buffer to create the final buffer. Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the ethanol.

  10-rxn kit 30-rxn kit
Wash Buffer 2 ml (entire bottle)5 ml (entire bottle)
100% Ethanol 8 ml 20 ml
Final Volume 10 ml 25 ml

Store the Wash Buffer prepared with ethanol at room temperature.
 
Fluorescent dyes are sensitive to photobleaching. Be careful to minimize exposure of the dye solution to light. The dye coupling reaction must be incubated in the dark. DMSO (used to resuspend the dyes) is hygroscopic and will absorb moisture from the air. Water absorbed from the air will react with the NHS ester of the dye and significantly reduce the coupling reaction efficiency.Keep the DMSO supplied in the kit in an amber screw-capped vial at –20°C, and let the vial warm to room temperature before opening to prevent condensation.
 
First-Strand cDNA Synthesis

The following procedure is designed to convert 5–20 µg of purified total RNA 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) in place of the total RNA in Step 2.
 
  1. Mix and briefly centrifuge each component before use.

  2. Prepare each reaction as follows in a 1.5-ml RNase-free tube:
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    Component Volume
    5–20 µg purified total RNA X µl
    Anchored oligo(dT)20 primer (2.5 µg/µl) 2 µl
    DEPC-treated water to 18 µl

     
  4. Incubate tubes at 70°C for 5 minutes, and then place on ice for at least 1 minute.

  5. Add the following to each tube on ice:

    Component Volume
    5X First-Strand buffer 6 µl
    0.1 M DTT 1.5 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

  6. 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.

 
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.
 
Purifying the First-Strand cDNA

Use the following procedure to purify the first-strand cDNA.
 
  1. Add 700 µl of Binding Buffer (prepared with isopropanol as described on page 14) to the reaction tube containing the first-strand cDNA from Hydrolysis and Neutralization, Step 3, previous page. Vortex briefly to mix.

  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 × 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) as described on page 14) 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 first-strand cDNA in the amber tube. The eluate contains your purified cDNA.   Proceed directly to Coupling the Fluorescent Dye below.
 
Coupling the Fluorescent Dye

Follow the steps below to couple Alexa Fluor® dye to the amino-modified first-strand cDNA. Use only the DMSO provided with this kit.
 
  1. Dry the purified first-strand cDNA from Step 9, above, in a speed vac at medium heat until the volume is reduced to ~3 µl. Be careful not to overdry the sample.

  2. Add 5 µl of 2X Coupling Buffer to the tube 

  3. Add 2 µl of DMSO directly to a vial of Alexa Fluor® Reactive Dye to resuspend the dye. Vortex thoroughly and then spin briefly to collect the contents.

  4. Add the DMSO/dye solution to the tube from Step 2 and vortex to mix thoroughly.

  5. Incubate the tube at room temperature in the dark for 1–2 hours. The reaction can be stored overnight if necessary.
 
Purifying the Labeled cDNA

Use the following procedure to purify the fluorescently labeled cDNA.
 
  1. Add 700 µl of Binding Buffer (prepared with isopropanol) as described on page 14) to the reaction tube containing the labeled cDNA from Coupling Procedure, Step 5. Vortex briefly to mix.

  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 × 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) as described on page 14) 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 cDNA. The eluate contains your purified labeled cDNA.
 
The sample can be 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.
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Direct cDNA Labeling

Introduction

This section provides protocols for using the SuperScript™ Plus Direct cDNA Labeling System, included with BioModule™ catalog no. WFGE04.
Using this system, you synthesize first-strand cDNA from total RNA using fluorescently labeled nucleotides in a reaction with SuperScript™ III Reverse Transcriptase. After cDNA synthesis, the RNA template is hydrolyzed, a purification step removes any unincorporated nucleotides, and the fluorescently labeled cDNA is ready for hybridization to microarrays.
 
Additional Materials Needed

In addition to the SuperScript™ Direct cDNA Labeling System components, you will need the following items:

  • Vortex mixer
  • Microcentrifuge
  • Water bath, incubator, or thermal cycler
  • Aerosol resistant RNase-free pipette tips
  • 0.5-ml or 1.5-ml RNase-free microcentrifuge tubes
  • 0.1 N NaOH
  • 0.1 N HCl
  • 100% Isopropanol
  • 100% Ethanol

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.
 
Amount of RNA

The kit has been optimized for use with 5–20 µg of total RNA as starting material. Lower amounts of starting material may be used, but may result in lower hybridization signals.
 
Alexa Fluor® 555 and Alexa Fluor® 647-labeled Nucleotides

The Alexa Fluor® 555 and Alexa Fluor® 647 dyes used to label the nucleotides are compatible with commonly used microarray scanners. 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
 
5X First-Strand Buffer

The 5X First-Strand Buffer includes 250 mM Tris-HCl (pH 8.3 at room temperature), 375 mM KCl, and 15 mM MgCl2.
 
Preparing Binding Buffer with Isopropanol

The Binding Buffer supplied with the Purification Module must be mixed with 100% isopropanol prior to use. Add the amount of isopropanol indicated below directly to the bottle of Binding Buffer to create the final buffer. Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the isopropanol.
 
                                             10-rxn kit                                      30-rxn kit
Binding Buffer                      5.5 ml (entire bottle)                     18.0 ml (entire bottle)        
100% Isopropanol                2.0 ml                                             6.5 ml
Final Volume                         7.5 ml                                            24.5 ml
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Store the Binding Buffer prepared with isopropanol at room temperature.
 
Preparing Wash Buffer with Ethanol

The Wash Buffer supplied with the Purification Module must be mixed with 100% ethanol prior to use. Add the amount of ethanol indicated below directly to the bottle of Wash Buffer to create the final buffer. Be sure to mark the appropriate checkbox on the bottle to indicate that you have added the ethanol.
 
                                        10-rxn kit                                            30-rxn kit
Wash Buffer                    2 ml (entire bottle)                               5 ml (entire bottle)        
100% Ethanol                   8 ml                                                     20 ml
Final Volume                   10 ml                                                     25 ml
 
Store the Wash Buffer prepared with ethanol at room temperature.

Fluorescent dyes are sensitive to photobleaching.  Be careful to minimize exposure of the labeled nucleotides.  The dye coupling reaction must be incubated in the dark.
 

First-Strand cDNA Synthesis

The following procedure is designed to convert 5–20 µg of total RNA into labeled first-strand cDNA. Lower amounts of starting material may be used, but may result in lower hybridization signals.
 
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. In a 1.5-ml or 0.5-ml RNase-free tube, add the following:
  3. TOP
     
    Component                                                                                Volume
    5–20 µg total RNA                                                                           X µl
    Anchored oligo(dT)20 primer (2.5 µg/µl)                                         2 µl
    DEPC-treated water                                                                  to 15 µl
     
  4. Incubate tube at 70°C for 10 minutes, and then place on ice for at least 1 minute.

  5. Add the following to the tube on ice:
     
    Component                                                                                                                                  Volume
    5X First-Strand buffer                                                                                                                        6 µl
    0.1 M DTT                                                                                                                                           3 µl
    10X Nucleotide Mix with Alexa Fluor® 555-aha-dUTP or
    10X Nucleotide Mix with Alexa Fluor® 647-aha-dUTP                                                                      3 µl
    RNaseOUT™ (40 U/µl)                                                                                                                       1 µl
    SuperScript™ III RT (400 U/µl)                                                                                                           2 µl
    Final Volume                                                                                                                                     30 µl
     

  6. Mix gently and collect the contents of each tube by brief centrifugation. Note:   After addition of the labeled nucleotides, be careful to minimize exposure of the tube to light.

  7. Incubate tube at 46°C in the dark for 3 hours. Note:   A 2-hour incubation is sufficient for generating high-quality labeled cDNA with high levels of picomole incorporation; however, a 3-hour incubation will result in 10–20% greater incorporation of labeled nucleotides and more full-length cDNA.

 
After incubation, proceed directly to Hydrolysis and Neutralization, below.

Hydrolysis and Neutralization

After cDNA synthesis, immediately perform the following hydrolysis reaction to degrade the original RNA:
 
  1. Add 15 µl of 0.1 N NaOH to each reaction tube from Step 6, above. Mix thoroughly.

  2. Incubate tube at 70°C for 30 minutes.

  3. Add 15 µl of 0.1 N HCl to neutralize the pH and mix gently.
 
Purifying the Labeled cDNA

Use the following procedure to purify the labeled cDNA.
 
  1. Add 700 µl of Binding Buffer (prepared with isopropanol) as described on page 19) to the reaction tube containing the labeled cDNA from Hydrolysis and Neutralization, Step 3.

  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 × 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 be 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.
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Assessing Labeling Efficiency

Introduction

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 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 before scanning, as any unincorporated dye 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) × 37 ng/µl × volume in µl

  5. Calculate the amount of fluorescent dye using the following formulas:
     
    Alexa Fluor® 555 (pmole) = (A555–A650)/0.15 × volume in µl
    Alexa Fluor® 647 (pmole) = (A650–A750)/0.24 × 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) × 0.04]] × 150,000/(A555 – A650) × 8,919
     
    Base/dye ratio for Alexa Fluor® 647 =
    [(A260 – A320) – [(A650 – A750) × 0]] × 239,000/(A650 – A750) × 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 prepared a control reaction using 10 µg of Control HeLa RNA as starting material, the following amounts are expected.
 
Indirect Labeling:
 

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 Labeled cDNA                 Incorporated Dye                    Dyes Molecules/100 Bases
 ≥ 250 ng                             ≥ 24 pmole                                             ≥ 2.50
 
Direct Labeling:
 
Labeled cDNA             Incorporated Labeled Nuc.           Dyes Molecules/100 Bases
    ≥ 400 ng                             ≥ 30 pmole                                              ≥ 1.0
 
If you do not obtain these amounts, see Troubleshooting.
 
Because of the high purity of the cDNA from the Low-Elution Volume Spin Cartridges, the yield and picomole dye incorporation calculations will be more accurate than with other purification methods. 




In the 1.2% E-Gel above, 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.


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Microarray Hybridization & Wash

Introduction

After cDNA labeling and purification, you are ready to hybridize your samples to a glass-slide microarray. This section provides protocols for hybridization using the BioModule™ Microarray Hybridization Solutions.
 
Amount of Labeled cDNA

For hybridization, use the entire volume of the labeling reaction from Step 9, (Indirect Labeling), or Step 9,  (Direct Labeling).

Glass Slide Surface Chemistries

The BioModule™ Microarray Hybridization Solutions have been developed and optimized for use with Corning® GAPS, GAPS II, and UltraGAPS™ slides.
 
Types of Capture Probes

The protocol in this manual produces good results with oligonucleotide probes of approximately 70 bases and longer.
 
MEEBO (Mouse Exonic Evidence-Based Oligonucleotide) and HEEBO (Human Exonic Evidence-Based Oligonucleotide) probe sets are collections of open source 70-mer oligo probes, largely derived from constitutively expressed exons, allowing interrogation of thousands of genes. These sets are available separately from Invitrogen.
 
Avoiding Background on Slides

Take care when processing slides to avoid contamination by dust particles or residue from dried droplets of buffers on either side of the glass slide. Any contamination of this sort can cause nonspecific background. To minimize background:

  • Make sure that buffers do not air dry on the slides between or after washes.
  • Immediately centrifuge or use clean, filtered, compressed air to dry the slides after pre-hybridization and washing.
  • Never use powdered gloves. Handle slides with forceps if possible, especially during the final wash step.

Blocking Non-Specific Binding

For customers spotting their own arrays on Corning® GAPS, GAPS II, and UltraGAPS™ slides, chemical blocking with succinic anhydride is not recommended because it can lead to high levels of non-specific background. The pre-hybridization procedure described is sufficient to block nonspecific binding of target cDNA to the slide surface. In addition, we do not recommend washing the slides with SDS-containing buffers after printing and prior to processing of the arrays.
 
Negative Controls

For negative control spots, we recommend using DNA from a species different and distinct from the one studied. Spotting buffer, such as 3X SSC, is not an appropriate negative control because it may produce a higher background.


Additional Materials Needed
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In addition to the components in this unit, you will need the following items:

  • Microarray—PCR product or oligonucleotide probes printed on Corning® GAPS, GAPS II, or UltraGAPS™ Slides
  • Slide box, or clean, dry slide mailer(s)
  • Hybridization chamber (e.g., Corning® Hybridization Chamber, catalog # 2551 or # 40080)
  • Raised-edge coverslips (e.g., LifterSlips™, Erie Scientific, catalog # 22x50I-2-4711 or 22x60I-2-4861)
  • Hybridization blocking DNA (e.g., Human Cot-1 DNA®, Mouse Cot-1 DNA®, or Salmon-Sperm DNA Solution)   Digital microarray scanner (e.g., the GenePix® 4000B from Molecular Devices) and associated software
  • Slide rack
  • Wash containers for individual slides (e.g., Coplin jars)
  • Wash tanks, capable of completely submerging a slide rack
  • Vortex mixer
  • Orbital shaker
  • Microcentrifuge
  • Heat block (95°C)
  • Water bath (42°C)
  • Temperature-controlled incubator (42°C)
  • Tabletop centrifuge with a microtiter plate rotor adapter capable of holding a slide rack or slide holder or filtered, compressed air
  • Lint-free laboratory wipes
  • Squirt Bottle
  • Aerosol-resistant pipette tips
  • 1.5-ml RNase-free microcentrifuge tubes

Prepare the following solutions before use. Solutions should be used within a few days after preparation.
Warm Buffer WB to 42°C to ensure that any precipitate has dissolved.
 
Pre-Hybridization Solution

The Pre-Hybridization Solution is supplied as a concentrate. To prepare for use, add Buffer WB and formamide directly to the bottle, as follows:
 
Pre-Hybridization Solution                                            129.5 ml (entire bottle)
Buffer WB                                                                     1.75 ml
Formamide (molecular biology grade)                           44 ml
 
Store at 2–8°C, and heat to 42°C before each use to ensure that any precipitate is completely dissolved.

Hybridization Solution

The Hybridization Solution is supplied as a concentrate. To prepare for use, add Buffer WB and formamide directly to the bottle, as follows:
 
Hybridization Solution                                                         720 µl (entire bottle)
Buffer WB                                                                           30 µl
Formamide (molecular biology grade)                                 750 µl
 
Store at 2–8°C, and heat to 42°C before each use to ensure that any precipitate is completely dissolved.
 
Wash Solution 1

Prepare 700 ml of Wash Solution 1 to process 3–4 slides. Prepare as follows:
 
  1.     Combine the following reagents in an appropriate container, in the order they are listed.
 
De-ionized water                                                           623 ml
Buffer WA                                                                       70 ml
Buffer WB                                                                         7 ml
Total Volume                                                                  700 ml
 
  2.     Invert gently 3–5 times.
 
Store Wash Solution 1 at 2–8°C, and heat to 42°C before each use to ensure that any precipitate is completely dissolved.
 
Wash Solution 2

Prepare 400 ml of Wash Solution 2 to process 3–4 slides. Prepare as follows:
 
  1.     Combine the following reagents in an appropriate container, in the order they are listed.
 
De-ionized water                                                          394 ml
Buffer WA                                                                        2 ml
Buffer WB                                                                        4 ml
Total Volume                                                                 400 ml
 
  2.     Invert gently 3–5 times.
 
Keep Wash Solution 2 at room temperature.
 
Wash Solution 3

Prepare 1,000 ml of Wash Solution 3 to process 3–4 slides. Prepare as follows:
 
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  1.     Combine the following reagents in an appropriate container, in the order they are listed.
 
De-ionized water                                                           995 ml
Buffer WA                                                                         5 ml
Total Volume                                                               1,000 ml
 
  2.     Invert gently 3–5 times.
 
Keep Wash Solution 3 at room temperature.Continued on next pageMicroarray Hybridization, continued
Pre-Hybridization Procedure

Follow the procedure below to prepare your slides for hybridization.
 
  1. Pre-heat the Pre-Hybridization Solution (prepared with Buffer WB and formamide as described)  on page 25) to 42°C. Place the printed slides in a polypropylene slide mailer, and fill the mailer with enough pre-heated Pre-Hybridization Solution to completely submerge the slides.

  2. Close the slide mailer, and invert gently 3–5 times.

  3. Incubate the slide mailer at 42°C for 30 minutes. During the incubation, fill the wash containers as described in the next step, and you can begin preparing the hybridization mixture as described below.

  4. Fill 5–6 wash containers (e.g., Coplin jars) with de-ionized water at room temperature (15–25°C). Process each slide individually in steps 5–8.
  5.  
     Note:   Replace the de-ionized water in the containers after processing five slides.
  6. At the end of the 42°C incubation, remove a single slide from the slide mailer. Submerge it in the first wash container and agitate for 5 seconds.

  7. Transfer the slide immediately to the next wash container and agitate for 5 seconds. To avoid background spots, do not allow the liquid to dry on the slide between washes.

  8. Repeat Step 6 until you have washed the slide once in each of the 5–6 wash containers.

  9. To dry the slide, use one of the following methods (there should be no residue from the Pre-Hybridization Solution remaining on the dried slide):

    • Prepare a centrifuge with a microtiter plate rotor adapter that will accept the slide rack containing the array slide. (Balance the opposing arm of the rotor with a slide rack containing an equivalent number of empty slides.) Quickly transfer the slide rack with the slide to the centrifuge, and immediately spin for 2–4 minutes at 600 × g to dry. Do not centrifuge at higher speeds, or the slide might break.
    • Dry the slide under a stream of clean, filtered, compressed air. Hold the slide with the barcode at the bottom, and direct the stream of air from top to bottom, perpendicular to the slide surface. Let the water flow off smoothly and avoid streaks.


  10. Repeat steps 5–8 for each slide. After all the slides are washed and dried, proceed to Preparing the Hybridization Mixture.

 
The volumes in the hybridization protocol are calculated for use with LifterSlips™ with dimensions of 25 × 60 mm. If you are using a different size of cover slip or LifterSlip™, you may adjust the volumes accordingly.
 
Blocking DNA (e.g., Human Cot-1 DNA®) supplied at 1 mg/ml should be concentrated to 10 mg/ml using ethanol precipitation prior to adding it directly to the hybridization reaction. See the documentation provided with the blocking agent for more information.
 
In our experience, the final signal intensity depends on the total amount of labeled cDNA used for hybridization, not on the cDNA concentration. Different hybridization volumes will not significantly affect the intensity.Continued on next pageMicroarray Hybridization, continued
 
 
During the following hybridization and post-hybridization procedures, be careful to minimize exposure of the labeled cDNA and array to direct light, to avoid photobleaching.
Make sure that your hybridization incubator thermometer is calibrated.

Always wear powder-free latex gloves when handling arrays. Avoid contact with the printed array surface.

 
Prepare the hybridization mixture for each slide as follows.
 
Note: For a single-color hybridization, use the entire volume of the labeling reaction (~16 µl) in the hybridization mixture. For a dual-color hybridization, use the entire volume from both labeling reactions (~32 µl).

  1. Warm the Hybridization Solution (prepared with buffer and formamide as described) to 42°C before starting, and ensure that any precipitate is completely dissolved.

  2. Prepare a hybridization mixture as follows:
  3.  
    Component                                                                                                                 Volume per slide
    Hybridization Solution                                                                                                               42 µl
    Hybridization Blocking DNA, 10 mg/ml (e.g., Human Cot-1 DNA®,
    Mouse Cot-1 DNA®, Salmon-Sperm DNA)                                                                               1 µl
    Labeled cDNA (for dual-color hybridizations, combine both
    labeling reactions as specified above)                                                                               ~16/32 µl
    De-ionized Water                                                                                                                   to 84.0 µl
     
     
  4. Gently vortex and heat the hybridization mixture at 95°C for 5 minutes. Centrifuge the tube briefly to bring down any condensation.

 
Hybridization Procedure

Follow the procedure below to hybridize your target DNA to the slides. Process each slide individually.
 
Place a slide prepared with Pre-Hybridization Solution (Step 9) flat with the array facing up in an open, clean, dry hybridization chamber. Place a clean, dust-free LifterSlip™ over the array area of the slide.
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Troubleshooting

Problem
Possible Cause
Solution
Total RNA Purification
Low RNA yield
Incomplete lysis
  • Decrease the amount of starting material used or increase the volume of TRIzol® to achieve complete lysis. The volume of tissue should not exceed 10% of the volume of TRIzol® Reagent used for homogenization.
  • Cut tissue samples into smaller pieces and ensure the tissue is completely immersed in TRIzol® Reagent.
 
Poor quality of starting material
  • The yield and quality of RNA depends on the type and age of the starting material.
  • Be sure to use fresh sample and process immediately after collection or freeze the sample at –80°C or in liquid nitrogen immediately after harvesting.
 
RNA precipitated out of solution after adding ethanol to TRIzol® preparation
Add ethanol in sequential volumes as specified and mix carefully between additions.
 
Suboptimal elution conditions
  • Be sure to incubate for 1 minute before elution.
  • To recover more RNA, perform a second elution step.
Low RNA yield, continued
Improper handling of sample from harvest until lysis
  • If not processed immediately, quick-freeze tissue immediately after harvesting and store at –80°C or in liquid nitrogen.
  • Frozen samples must remain frozen until TRIzol® Reagent is added.
  • Homogenize tissues quickly after adding TRIzol® Reagent.
 
Tissue very rich in RNases (e.g. pancreas)
RNA isolated from tissue rich in RNases may require the addition of RNAse inhibitors/inactivators to protect the RNA from degradation.
 
RNA has been damaged or degraded
Re-purify RNA; follow the specified RNA handling guidelines and be careful to maintain aseptic conditions.
Total RNA Purification, continued
Low A260 ratio
Sample was diluted in water; non-buffered water has variable pH (4)
Use 10 mM Tris-HCl (pH 7.5) to dilute sample for OD measurements.
28S and 18S bands are not observed after agarose gel electrophoresis
Too little RNA loaded on the gel
Be sure to load at least 250 ng of RNA for analysis.
 
RNA has been damaged or degraded
Re-purify RNA; follow the specified RNA handling guidelines and be careful to maintain aseptic conditions.
cDNA Labeling
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 5 µg of total RNA.
 
Poor quality RNA used or RNA is degraded
Check the quality of your RNA preparation on a gel. 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.
 
Concentration of NaOH and/or HCl used in the hydrolysis and neutralization reaction is incorrect. This affects the pH of the reaction and therefore may affect binding to the column.
Verify the concentration of NaOH and HCl, and repeat the reaction if necessary.
 
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 fluorescent dye/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 dyes and 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.
Microarray Hybridization
No signal or weak signal on the microarray
Hybridization or wash temperature too high
Check the temperature with a calibrated thermometer during hybridization and washing. If your array contains short oligonucleotides (<60 bases), it may be necessary to use a lower temperature for hybridization and/or washing.
 
Hybridization time too short
Incubate the slide at 42°C for at least 16 hours.
 
Condensation on slide surface during hybridization
Use individual hybridization chambers for each hybridization. Do not add more liquid to the chamber than recommended by the manufacturer.
 
Photobleaching of the Alexa Fluor® dyes
Avoid direct exposure of the dyes, labeled cDNA, and hybridized array to light. Perform hybridization and wash procedures in low light conditions.
 
Array slide scanned in wrong orientation
Check the position of the slide in the scanner; reposition and rescan if necessary.
Coverslip stuck to array surface
Hybridization chamber not properly sealed or humidified
Make sure that the chamber is properly sealed with the correct amount of liquid prior to incubation.
 
Inadequate volume of Hybridization Solution used for coverslip size
Make sure that the Hybridization Solution completely covers the array surface under the LifterSlip/coverslip.
High or uneven background on the array
Residual wash solutions dried on microarray slide
Transfer the slide quickly between wash containers, and centrifuge immediately after the final wash step. Avoid exposing the slide to air between washes for more than a few seconds. Dried wash solution will appear as streaks on the slide.
 
Dehydration of the hybridization mixture
This frequently appears as high background around the edges of the LifterSlip/coverslip. Make sure that the hybridization mixture completely covers the array surface under the LifterSlip/coverslip, and that humidity is maintained during incubation.
 
Air bubbles trapped under the LifterSlip during hybridization
Air bubbles can prevent the target from coming into contact with the spotted probe. Make sure that LifterSlips are clean and dust-free before applying them to the slide. Small air bubbles may dissipate during hybridization.
 
Flat cover slips used
Flat cover slips do not allow sufficient volume for hybridization. Use a LifterSlip or a cover slip with lifter bars on the sides to accommodate additional liquid volume.
 
Scratches from LifterSlip
Do not try to move the LifterSlip after placing it on the slide. During the initial washing step after hybridization the LifterSlip should glide off the slide easily. Do not attempt to remove the LifterSlip manually.
 
Improper array handling
Always wear powder-free gloves when handling the array, and avoid touching the slide surface.
 
Poor slide quality
Arrays scanned prior to hybridization should show no fluorescence. Scan a slide from each printing batch prior to hybridization.
 
Canned air was used to dry the slide
Canned air contains propellants, which can leave a visible residue and reduce the image quality. Use a clean, filtered air source or centrifugation to dry the slides.
Microarray Hybridization, continued
Snowy, flocculent background on the array
Residue from pre-hybridization carried over into wash tanks
Do not use the wash tanks for pre-hybridization. Use slide mailers that are dedicated to these steps.
Nonspecific signals on the array
No competitor DNA in hybridization mixture
Add salmon-sperm DNA to the hybridization mixture as indicated in the procedure.
 
Hybridization time was too long
Decrease hybridization time. Hybridize for a maximum of 20 hours.
 
Hybridization temperature was too low
Perform hybridization at 42°C as described in the procedure. Check the temperature with a calibrated thermometer during hybridization and washing.
 
Incomplete washing after hybridization
Make sure that the wash solutions were prepared properlyand that all wash steps were performed as described.
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References

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

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

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

  4. Gerard, G. F., D'Alessio, J. M., Kotewicz, M. L., and Noon, M. C. (1986) Influence on stability in Escherichia coli of the carboxy-terminal structure of cloned Moloney murine leukemia virus reverse transcriptase. DNA, 5, 271-279

  5. Kotewicz, M. L., D'Alessio, J. M., Driftmier, K. M., Blodgett, K. P., and Gerard, G. F. (1985) Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene, 35, 249-258
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LT064