SPIA cDNA

Ovation Biotin RNA Amplification and Labeiing System Purification of SPIA cDNA... 

Pipet 25 pL of the cleaned SPIA cDNA into a clean 0.2-mL mbe. 

Ovation Biotin RNA Ampiification and Labeiing System Biotin Labeiing of SPiA cDNA... 

Add 5 pL of labeling buffer to each fragmented SPIA cDNA reaction and mix by triturating. 

Carefully spot the labeled SPIA cDNA (final volume is 50pL) onto the center of the column being careful not to touch the resin (see Note 44). 

Remove and discard the spin column. The eluate (final volume is 50-52pL) contains the labeled SPIA cDNA. 

The yield and purity is determined using spectrophotometry at OD g. The spec-trophometer should be set to calculate the concentration based on the absorbance of single-stranded DNA (the SPIA product is antisense single-stranded cDNA). For yields around 7pg eluted in 50pL the concentration ise 140ng cDNA/pL. The biotin-labeled SPIA cDNA can be stored at -20°C. 

Ovation Biotin RNA Amplification and Labeling System Hybridization of Biotin-Labeled SPIA cDNA to a GeneChip... 

The purified biotin-labeled SPIA cDNA is now ready to be hybridized to a GeneChip. The labeled extract generated here can now be treated in exactly the same manner as that described for the ENZO-generated product, with the excep-... 

When loading the column, the labeled SPIA cDNA must be placed directly on the center and not allowed to pass down the side and into the collection tube. 

Transfer each SPIA reaction to a sterile 1.5-mL tube on ice and add 600pL of cDNA binding buffer to 150 oL of the SPIA amplified cDNA reaction. Mix by vortexing for 3 sec and pulse in a centrifuge to collect. 

The expected yield of the SPIA amplification reaction is approximately 7pg (although can be anywhere between 5 and lOpg) from lOng of total RNA in an initial volume of 160 pL. The analysis of 4pL of the SPIA reaction volume should provide >200ng amplified cDNA product to be visuahzed on an agarose gel. If an amplification product cannot be seen from this volume of the reaction, it is likely... 

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