Custom GeneDetect® ONE-STEP RNA probe synthesis templates (choice of T7, T3 or SP6 OptiScript^TM promoters). 

"It is now possible to prepare an optimized RNA hybridization probe (riboprobe) to any target gene cheaply and faster than ever before".

Fig 1. Preparation of an RNA probe for in situ hybridization by in vitro transcription.

Single-stranded RNA probes also called complementary RNA (cRNA) or riboprobes (it must be noted that riboprobe^Ò is actually a registered trademark of Promega Corporation) are often used for in situ hybridization because: 

1. they are extremely sensitive (due to the fact that they can be labeled to high specific activity during probe synthesis) 

2. RNA-RNA hybrids are more stable than DNA-RNA hybrids and 

3. non-specific tissue signals can be removed after hybridization with RNase A since RNA duplexes (representing specific binding) are resistant to degradation by RNase A. 

One major disadvantage of using RNA probes is the amount of effort required to actually prepare these probes.

RNA probes are usually prepared by in vitro transcription (see Fig. 1 above). The RNA probe is transcribed from a linear DNA template using highly specific bacteriophage DNA-dependant RNA polymerases from the Salmonella bacteriophage SP6, and the E. coli. bacteriophages T3 and T7 (RNA polymerase T7, T3 or SP6). The investigator must therefore obtain sufficient quantities of a plasmid carrying the gene sequence of interest that can be used as the template for RNA probe synthesis. Furthermore, before a riboprobe can be transcribed the correct RNA polymerase promoter sequences must be available in the plasmid in the correct orientation with respect to the template sequence. If the cloned gene exists in a plasmid lacking these promoter regions the investigator is forced to subclone the gene into a more suitable vector. For example the transcription vectors pGEM (SP6 and T7 promoters, Promega) and pBluescript (T3 and T7 promoters, Stratagene) are commonly used. For in vitro transcription reactions the plasmid must also be in a linear form. The investigator must use restriction enzymes to linearize the plasmid. Even after the RNA probe has been transcribed successfully from the template, if the RNA probe is greater than 300-400bps in length it should be hydrolysed into shorter fragments since the optimal upper length for riboprobes for in situ hybridization is 150-200bps. Longer probes have poor tissue penetration. Lastly, and perhaps most limiting is the possibility that the investigator may not be able to easily source the cloned gene sequence required for RNA probe generation.

We have attempted to resolve these issues by making available our ONE-STEP RNA probe synthesis templates. Using our custom designed templates it is now possible to prepare an optimized RNA hybridization probe to any target gene cheaply and faster than ever before.


To order.

1. Submit order details 

How supplied.

Example specification sheet

You are supplied with 50µg of a 100mer PAGE purified double stranded DNA template that can be used for the in vitro transcription of an 80mer riboprobe^Ò  using any commercial riboprobe^Ò  synthesis kit.

GeneDetect® ONE-STEP RNA probe synthesis templates come standard with an OptiScript^TM promoter sequence for T7 RNA polymerase. The standard T7 promoter has been redesigned to produce the GeneDetect® OptiScript^TM promoter which allows for optimal riboprobe^Ò  generation by limiting premature abortive transcription. You can specify to replace the T7 OptiScript^TM promoter with another promoter (either OptiScript^TM T3 or SP6) when purchasing. 

Pricing. Does not include shipping or handling.

Contact Sales@GeneDetect.com for more information.

NB: Riboprobe^Ò  is a registered trademark of Promega Corporation.