The DFCI Microarray Core offers two different processing methods, referred to below as Affymetrix process or NuGEN process, to covert RNA into biotinylated cRNA or cDNA, respectively, either of which is suitable for hybridization to the 3’ arrays:

• Affymetrix processing requires 5-15 µg of RNA and is the method used in the Core since the Core’s inception in 2002.  The procedure involves the synthesis of double-stranded cDNA which then serves as a template for the synthesis of biotinylated cRNA by in vitro transcription.  The cRNA is hybridized to the 3’ arrays. 
• The NuGEN Ovation V2 Amplification System has recently been added to the Core’s offerings.  This procedure is a cDNA synthesis followed by an isothermal linear displacement amplification reaction to produce anti-sense single stranded cDNA that can be hybridized to Affymetrix 3’ gene expression arrays.  It is a linear amplification service that can start with as little as 5ng of total RNA.
  

RNA Isolation

The analysis in the Core begins with total RNA that is provided by an investigator. In our experience, RNA prepared using the Trizol reagent followed by additional purification using RNeasy from Qiagen yields RNA that produces satisfactory microarray data. Modifications of those methods, as well as other methods are also known to produce suitable RNA. Please inquire if you are uncertain as to whether an RNA extraction method that you are considering is suitable.

RNA Amount for Affymetrix Processing

Investigators are asked to provide 15 µg of each RNA sample, if possible. If it is not possible to provide 15 µg of RNA, a smaller amount will often be suitable.  For most specimens, 5 - 15 µg of RNA has been found to consistently produce excellent data. Results from analyses that begin with 1 - 4 µg of RNA are more likely to fail, but in our hands, approximately half of the specimens that begin with this amount produce excellent results. Ideally, the RNA should be in a volume of 10 µl (maximum volume accepted is 50uL).

RNA Amount for NuGEN Ovation V2 Processing

Please provide 300 ng in a total volume of 8 µl.

Quality Control

Two quality control measures are carried out on a small aliquot of the submitted RNA samples: (1) a spectrophotometric analysis to confirm the concentration and to detect contaminating proteins and other molecules, and (2) a size fractionation procedure using a capillary electrophoresis instrument (Bioanalyzer 2100 from Agilent Technologies) to determine whether the RNA is intact. An investigator will be notified if one or more of his/her samples are deemed unsuitable for analysis.

3' Arrays

The oligonucleotide arrays used in the Microarray Core are manufactured by Affymetrix. The manufacturing process involves the synthesis of 11 different perfect match (PM) and 11 different single base mismatch (MM) 25-mers per gene; the 25-mer probes are synthesized in situ using photolithography and combinatorial chemistry. Additional information on the manufacturing process and on probe selection, probe arraying and quality control is available from the Affymetrix website.

Affymetrix Laboratory Process –

1. RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out.
2. The double-stranded cDNA is used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates.
3. The biotinylated complementary RNA (cRNA) is purified from the IVT reaction mixture using the RNeasy system (Qiagen). The cRNA is quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. Those cRNA's that fall outside of an acceptable range will not be carried forward in the analysis. Should this occur, investigators would be notified and asked to provide a new RNA sample.
4. The purified cRNA is fragmented in order to facilitate the subsequent hybridization step.
5. The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
6. The chips are then transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
7. Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. The data is transferred to a file-system from which it can be downloaded via a password-protected URL.
   

NuGEN Ovation V2 Laboratory Process –

1. First strand cDNA is prepared from total RNA using a DNA/RNA chimeric primer and a reverse transcriptase. Synthesis of a second strand results in a double stranded cDNA with a unique DNA/RNA heteroduplex at the 5’end of the antisense strand.
2. The products are amplified using SPIA™ amplification.  RNaseH degrades the RNA in the DNA/RNA heteroduplex.  A new SPIA™ DNA/RNA chimeric primer hybridizes to the exposed DNA sequence.  DNA polymerase initiates replication at the 3’ end of the primer, displacing the existing strand.  This process of SPIA™ DNA/RNA primer hybridization, DNA replication, strand displacement and RNA cleavage is repeated, resulting in rapid accumulation of cDNA with sequence complementary to the original RNA. 
3. The SPIA™ amplified cDNA is purified using the Zymo Research DNA Clean & Concentrator™ system. The cDNA is quantified spectrophotometrically.  Those cDNA's that fall outside of an acceptable range will not be carried forward in the analysis. Should this occur, investigators would be notified and asked to provide a new RNA sample.
4. The purified cDNA is fragmented through a chemical and enzymatic process.  The fragmented product is labeled via enzymatic attachment of a biotin-labeled nucleotide to the 3’-hydroxyl end of the fragmented cDNA.
5. The biotinylated cDNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
6. The chips are then transferred to a fluidics instrument that performs washes to remove cDNA that has not hybridized to its complementary oligonucleotide probe. The bound cDNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
7. Each cDNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. The data is transferred to a file-system from which it can be downloaded via a password-protected URL.