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5' END DISCOVERY PANELS

What is the advantage of performing a 24-tissue RACE analysis if the tissue site(s) of expression of my gene is known?
Can I use the Sure-RACETM panel to study 5 alternate RNA splicing in different tissues?
Can I use the RACE panel to determine the sites of expression of my gene?
Can the Sure-RACETM panel be used for 5 end to 3 end PCR amplification to generate a full- length clone?
Can I use the RACE products for direct sequencing?
How do I choose the right band for sequencing if multiple components appear in the same or different tissues after the RACE reaction?
Can I use the RACE panel to isolate a full-length clone if I have a 3-end EST sequence?
Can I use the Sure-RACETM panel for 3-end RACE?
Can I dilute the cDNAs in the RACE panel and use them for multiple experiments?
Can I transfer the cDNA from the panel to other types of PCR tubes that fit my thermal cycler?

What is the advantage of performing a 24-tissue RACE analysis if the tissue site(s) of expression of my gene is known?

By surveying more tissues where your gene is expressed, you markedly increase the probability of successfully completing the 5 end of your transcript of interest.  Each cDNA synthesis yields a different proportion of full-length reverse transcripts because of differences in the intactness of the starting mRNA, in relative mRNA accumulation between tissues, and in the efficiency of cDNA synthesis between RNA populations.  Additionally, the appearance of an identical PCR product in different tissues assures specificity of the RACE analysis.

Can I use the Sure-RACETM panel to study 5 alternate RNA splicing in different tissues?

Yes.  The 24-tissue RACE panel was specifically designed not only for standard RACE analysis, but also for studying 5-end differential splicing and alternate RNA start-sites.  Tissue-specific alternate transcriptional initiation sites, alternate intron splicing and exon usage, can be determined by using the Sure-RACETM panel with just two gene-specific primers.

Can I use the RACE panel to determine the sites of expression of my gene?

No.  We do not recommend using these panels to study tissue distribution of expression of your gene.  The amount of cDNA has not been normalized between the tissues.  As such, it can only give you a rough idea of where your gene is expressed but not the relative levels of expression between the various tissues.  OriGenes Rapid-ScanTM Gene Expression Panels are designed specifically for defining gene expression profiles.

Can the Sure-RACETM panel be used for 5 end to 3 end PCR amplification to generate a full- length clone?

Yes.  These cDNAs may be used for long cDNA cloning because of the strategy that was used for their synthesis. Full-length cDNAs as long as 13-kb in length have been detected in testing.  To generate long PCR products, you may have to choose the appropriate thermostable DNA polymerase (e.g. Taq) and prolong the extension time.

Can I use the RACE products for direct sequencing?

Yes.  Individual bands can be excised from the agarose gel, and the DNA can be extracted and used directly for sequencing (QIAquick PCR purification kit).  You own GSP2 primer is a better choice for direct sequencing to avoid different truncations at 5 when ADP2 is used.  If the amount of PCR product is insufficient, excised band(s) can be cut out and re-amplified with the ADP2 and GSP2 primers. Meanwhile, the DNA bands can also be cloned into a TA cloning vector for sequencing.

How do I choose the right band for sequencing if multiple components appear in the same or different tissues after the RACE reaction?

There are several scenarios to consider.  The first is that no differential splicing occurs at the 5 end of your RNA.  If your gene is expressed in more than one tissue, you should choose the largest common band from these tissues.  Always choose well-separated bands for sequencing. The second scenario is that alternate transcriptional initiation or splicing exists at the 5 end of the RNA, either in the same tissue or in different tissues.  Comparison of different size bands in all the tissues will enable you to determine how many alternatively initiated or spliced transcripts exist.  While truncated PCR products may exist in some tissues, differentially initiated or spliced transcripts have defined sizes and they can be found frequently in more than one tissue.  To confirm the existence of alternatively initiated or spliced products, duplicate RACE reactions should be conducted using the four plates that are provided.

Can I use the RACE panel to isolate a full-length clone if I have a 3-end EST sequence?

Yes.  RACE is the quickest approach to obtain 5 end information based on the availability of a short 3 end sequence.  If the cDNA is under 5 kb in length, you usually can have the full length sequence from just one RACE experiment.  However, if your cDNA is above 5 kb, we suggest that you use long PCR for your analysis.  Alternatively, additional RACE can be conducted to complete the whole sequence based on the sequence from a previous RACE experiment.

Can I use the Sure-RACETM panel for 3-end RACE?     

No.  This product was specifically designed for 5-end RACE because two unique adapter primers were specifically linked to the 5 end.  Primers could be designed from the 3-end linker sequence for 3-end RACE, but it may not be as efficient as the 5-end RACE.             

Can I dilute the cDNAs in the RACE panel and use them for multiple experiments?       

Two cDNA concentrations are provided to facilitate analysis of both abundant and rare transcripts.  They are designated 5X for analysis of most transcripts and 1X for highly-abundant transcripts.  These concentrations have been tested extensively to give a positive signal even for very rare transcripts (for example, 2 copies per million).  In general, RACE analysis with a higher concentration of cDNAs gives you more consistent results.  If the cDNA concentration is too low, or your primer specificity is not ideal, you may not be able to amplify specific but rare targets and may detect only nonspecific but abundant targets.  We recommend that you use the concentration provided.  If you know that your RNA is very abundant in a specific tissue, we suggest that you start by using the 1X concentration.

Can I transfer the cDNA from the panel to other types of PCR tubes that fit my thermal cycler?       

Yes.  If the 48-well panel does not fit your thermal cycler, the cDNA can be transferred to any appropriate type of PCR tubes.  First, add 10 ul of dH2O to each well, incubate for 30 min. at room temperature, and mix gently to dissolve the cDNA.  Second, transfer the cDNA from each well to a fresh tube, prepare and add 10 ul of 2X PCR premix for the first-round PCR.   

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