Translational research attempts to directly connect basic research to patient care. Clinicians collect tissue samples from their patient groups for diagnosis and often enough make it available for performing molecular biology experiments. Tissues obtained using needle biopsy samplings are frozen in the surgery room for making tissue sections for diagnosis. The remaining samples are then archived by storage in -80ºC freezers. Utilization of these samples for molecular biology experiments is greatly limited due to the suboptimal amount and quality of starting material resulting from lack of snap chilling. This greatly limits the feasibility of using these clinical samples for major studies involving real-time PCR or microarray experiments.
RampUP from Genisphere is the ultimate RNA amplification kit that enabled us to use such clinical samples for gene expression studies. This kit is for amplifying any RNA sample 10 pg to 10 ng, including difficult samples that are obtained by Laser Capture Microscopy. This kit provides a two-round amplification protocol that is easy and accurate and generates sense-strand RNA from all kinds of RNA samples, including partially degraded RNA and prokaryotic RNA. Since the RNA samples we obtained from the frozen clinical biopsies are very minimal in amount and were degraded to varying extents, we found this kit to be very useful.
The RampUP kit uses a proprietary Tandem RNA Polymerase Promoter (TRPP) technology for producing the sense-strand amplified RNA. This technology uses a tandem RNA polymerase promoter oligo that includes T7 and T3 RNA polymerase promoter sequences. So the in vitro transcript generated from first round of amplification using T7 RNA Polymerase will have a built-in T3 RNA Polymerase promoter sequence. The T3 promoter sequence is used in the second round of amplification, thus eliminating the entire promoter synthesis process. RampUP also results in higher yields from very low starting RNA and results in no 3’bias. Amplification begins by synthesizing cDNA from the 3’ end of the message. The ensuing round-one T7-amplification reaction initiates at the 3’ end of the cDNA (originally the 5’end of the RNA). The protocol is such that the amplified RNA will not contain any non-specific amplification products. The senseRNA generated can be used for hybridization on a variety of platforms, qRT-PCR and many other RNA expression assays. We used the amplified RNA for hybridization on glass microarrays and obtained reproducible results.
The unique technology adopted in this kit makes two-rounds of amplification simple and straight-forward. Moreover, it enables you to perform experiments using suboptimal quality and quantity of RNA. From one needle biopsy core, we generally obtained between 100 and 500 ng RNA. For amplification, we generally started our reaction with ~10 ng total RNA and ended up getting between 1.5 and 2 µg of amplified RNA, depending on the intactness of starting total RNA. Thus, we had more of the total RNA left for other confirmatory experiments.
In general I would say I am glad we found this kit as it actually enables us to use the thousands of archival samples stored in the pathology freezers after diagnosis.
RampUP RNA Amplification Kit From Genisphere
Simple protocol that generates high quality amplified RNA from suboptimal samples.
Only half of the total cDNA synthesized is used for the <i>in vitro</i> transcription. The reagents provided in the kit are not sufficient to utilize the full reaction.
The Bottom Line
A useful kit that helps pathologists utilize their archival clinical samples for molecular analysis.