CYSCRIBE™ First Strand cDNA Labeling Kit

Ordering Kit Contents Specifications Product Description and Protocol Protocol Booklet Key Features of the Protocol Features and Benefits Experimental Data Related Products FAQ

For preparing highly fluorescent first strand cDNAs for use as probes in microarray hybridization

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Specifications

Product description and protocol

The CyScribe first strand cDNA labeling kit provides optimised reagents and protocols to generate cDNA probes labeled with either Cy3 or Cy5 deoxynucleotides. These probes generate even and bright signal levels in microarray hybridization applications, which can be easily measured on microarray scanners optimised to detect Cy3 and Cy5 fluorescence. Even incorporation of both Cy3 and Cy5 with the CyScribe kit facilitates accurate analysis and minimizes experimental variation caused by uneven incorporation of labels.

Preparation of CyDye labeled first-strand cDNA with the CyScribe kit

Key features

• The CyScribe enzyme is a variant of MMLV reverse transcriptase that gives excellent yields of first strand cDNA. The labeling reaction, catalysed by CyScribe reverse transcriptase incorporates Cy3-dCTP, Cy5-dCTP, Cy3-dUTP or Cy5-dUTP into first strand cDNA.
• Two optimised nucleotide mixes are provided with the kit to be used with CyDye-dCTP and CyDye-dUTP, respectively. Equal incorporation of Cy3-dCTP, Cy5-dCTP, Cy3-dUTP and Cy5-dUTP can be achieved.
• Three alternative priming methods are offered in this kit.
• i) Priming with anchored oligo(dT) will direct the start of the synthesis of cDNA from the 5’ end of the polyA-tail. This priming method is especially suitable if the hybridization targets on the microarrays are derived from the 3’ ends of transcripts.
• ii) As an alternative, the random nonamers supplied with the kit can be used for priming the synthesis of first-strand cDNA. These random 9 mer oligonucleotides will anneal to their complimentary sequences and direct the synthesis of complimentary cDNA molecules along the length of transcripts. Priming cDNA synthesis with random nonamers will shorten the average length of transcripts, but will not detrimentally affect the use of these cDNA molecules as a hybridization probe.
• iii) The standard protocol provided with the CyScribe kit uses both labeling methods together to provide uniform coverage of transcripts in the CyDye-labeled cDNA.
• The kit has been developed for use with purified mRNA that is free from contaminating DNA, proteins or nucleotides. With the standard protocol,100 ng to 1 µg of mRNA can be used as a template for the synthesis of CyDye labeled first-strand cDNA. To label larger amounts of mRNA, multiple reactions should be used.
• Total RNA can be used with the kit.
• Included in the protocol are recommendations for probe purification and quantification and hybridization.
• Proprietary microarray hybridization buffer is also included to ensure low backgrounds.

Features and benefits

Experimental data to demonstrate the performance of the CyScribe First Strand cDNA Labeling Kit

The CyScribe First Strand cDNA Labeling Kit demonstrates significantly improved labeling results when directly compared with SuperScript II.

1. Incorporation of CyDye into cDNA: CyScribe vs SuperScript II

When labeling reactions were performed with CyScribe and Super Script II using equal amounts of CyDye deoxynucleotides, almost 1.8 times more CyDye could be incorporated into cDNA with CyScribe.

The incorporation of Cy3 and Cy5 labeled deoxynucleotides is more even with the CyScribe kit-(ratio of incorporation of Cy3 to Cy5-1:0.94 with CyScribe vs 1:0.75 with SuperScript II).

Both labeling systems gave similar ng yields of cDNA.

The incorporation of Cy3-dCTP by SuperScript II was set at 100% as the control. The incorporation of the CyDye deoxynucleotides are expressed relative to this

2. Signal intensity from microarray hybridization: CyScribe gives brighter signals than SuperScript II.

When equal amounts (quantified as nanograms) of cDNAs prepared with CyScribe and SuperScript II are hybridized to identical microarray slides, the signal intensity from the CyScribe probes was 1.7 and 4 times brighter for Cy3 and Cy5 signals respectively.

In identical conditions, a brighter signal is achieved with CyScribe than generated with SuperScript II.

The general background is lower with the probes generated with the CyScribe kit.

All cDNAs were prepared from the same mRNA, hence no differential gene expression was expected. The greener colour of the spots on the second slide is the (unwanted) result of the more efficient incorporation of one dye by that enzyme. On the CyScribe slide, more spots are discernible from background, demonstrating increased sensitivity in detection of rarer transcripts.

Microarray slide hybridised with probe labeled using CyScribe first strand cDNA labeling kit. 40 ng of both Cy3 and dCy5 labeled cDNAs was used.

Microarray slide hybridised with probe labeled with SuperScript II 40 ng of both Cy3 and Cy5 labeled cDNAs was used.

The above comparison was done using 1µl of CyDye deoxynucleotide in the reaction mixture. Up to 3 times as much dye (3µl per reaction) has been used to gain brighter signals with other RT enzymes- only this increased quantity of dye will give results as bright as using 1µl with CyScribe.

The higher incorporation of CyDye into cDNA increases the sensitivity of detecting rarer mRNAs in samples, this is highly desirable in microarray analysis as most biologically relevant genes are expressed at low levels in target tissues.

3. More accurate data can be generated using CyScribe First Strand cDNA Labeling Kit

Comparison of results generated with CyScribe and SuperScript II where labeled probes contain equal quantities of fluorescent dyes, demonstrating that the same general pattern of differential gene expression is seen with both probes. However, the CyScribe generated probes are more sensitive,allowing the detection of genes that are expressed at lower levels in the sample tissues, those which fail to give appreciable signal with the other enzymes.

The general non-specific background is lower with CyScribe generated probes as a result of an optimised purification protocol.

In the labeling reactions, more pmols of CyDye can be incorporated into total cDNA using CyScribe.

CyScribe gives increased amounts of probes that give brighter hybridization signals.

Microarray slide hybridized with Cy3 and Cy5 labeled probes generated with the CyScribe kit.Equal amount (quantified in pmols) of both probes were used.

Microarray slide hybridized with Cy3 and Cy5 probes labeled with SuperScript II. Equal amount (quantified in pmols) of both probes were used.

Both slides are shown with exactly the same contrast settings.

4. Even incorporation of labeled deoxynucleotides

CyScribe has been optimised for the incorporation of four different labeled deoxynucleotides into cDNA: Cy3-dCTP, Cy5-dCTP, Cy3-dUTP and Cy5-dUTP. All four deoxynucleotides are incorporated into cDNA with almost equal efficiency.

The incorporation of Cy3-dCTP in series 1 was set at 1. The other values are expressed relative to this.

Data from two separate experiments is shown. Labeling reactions were performed with control RNA and the total incorporation of CyDye into cDNA was measured. The CyScribe kit incorporates Cy3 and Cy5-deoxynucleotides equally efficiently into cDNA. Furthermore, there is little difference in the incorporation of CyDye into cDNA when CyDye-dCTP and -dUTP are used.

5. Different amounts of mRNA can be labeled with the CyScribe kit

Fluorescent first strand cDNA can be prepared starting with as little as 100 ng of mRNA. It is recommended that the amount of mRNA does not exceed 1 µg as the yield of cDNA is no longer proportional to the amount of starting material beyond 500 ng.

Total RNA can be labeled using the CyScribe kit. Using less than 25 µg of RNA is recommended per labeling reaction (assuming that 2% of total RNA is mRNA).

Labeling reactions were performed with different amounts of mRNA and the yield of fluorescent cDNA was quantified. Data shows that successful labeling reactions can be performed with 100 ng of m3.5RNA. However, increasing the amount of mRNA in the reactions beyond 500 ng of mRNA per reaction, no longer results in linear increases in the yield of cDNA.

6. Interpretation of microarray hybridization data

The CyScribe first strand cDNA labeling kit is designed for generating probes for dual colour differential gene expression analysis on microarrays. Although unprocessed data can demonstrate the presence of differentially expressed transcripts, careful normalization of quantified data is required to achieve meaningful results.

Close-up of a microarray slide hybridized with Cy3-labeled muscle cDNA and Cy5-labeled brain cDNA. Equal amounts of both cDNAs (pmol of dye) were hybridized together on a microarray slide containing immobilised gene targets.

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