StarFire® miRNA Detection Kit

miRNA StarFire® is a proprietary labeling system for generating radiolabeled oligo probes with 10-fold greater specific activity than traditional 5′-end labeling with polynucleotide kinase. It is based on 3′-end labeling through extension with DNA polymerase. This labeling method is particularly useful for probes used to identify low copy targets, such as small regulatory RNA, and for analysis of the expression of microRNA genes using northern blots. [1-3]

miRNA StarFire Complete Kit

$195.00 USD
  • Sufficient for 25 labeling reactions
  • Includes:
    • Exo-Klenow DNA Polymerase
    • StarFire 10X Buffer Mix
    • StarFire Stop Buffer
    • One PAGE-purified StarFire Custom Probe (up to 50 bases)
    • StarFire Universal Template

miRNA StarFire Refill Kit

$145.00 USD
  • Sufficient for 25 labeling reactions
  • Exo-Klenow DNA Polymerase
  • StarFire® 10X Buffer Mix
  • StarFire® Stop Buffer
  • StarFire® Universal Template


miRNA StarFire Universal Template

$350.00 USD
  • Sufficient for 300 labeling reactions
  • 0.5 OD (in a single tube)
  • HPLC Purified 
  • Quality Control checked by mass spectrometry and CE

miRNA StarFire Custom Probes (up to 50 bases)

$0.65 USD / Base

Desalted Only, or PAGE Purified



The development of miRNA StarFire® has provided a method for efficient oligonucleotide labeling, a substantial improvement in per molecule specific activities, and can be adapted for use with non-radioactive tags. This method uses a DNA polymerase catalyzed primer extension reaction to generate oligonucleotide probes with ten-fold greater per molecule specific activity with radiolabels [1] or non-radioactive tags.

Figure 1. The StarFire® oligonucleotide labeling protocol. (A) Two oligonucleotides are used in the reaction–a target-specific probe oligonucleotide and a universal template oligonucleotide. The oligonucleotides are annealed (B), labeled in a primer extension reaction by DNA polymerase (C) and unincorporated nucleotides are removed by gel filtration (D). [1]

References

  1. Use of high specific activity StarFire oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S. pombe. Behlke, M.A., Dames, S.A., McDonald, W.H., Gould, K.L., Devor, E.J., and Walder, J.A., Biotechniques, 29:892-897 (2000).
  2. Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and Broad-Complex gene activity. Sempere, L.F., Sokol, N.S., Dubrovsky, E.B., Berger, E.M., Ambros, V., Developmental Biology 259: 9-18 (2003).
  3. The Expression of the let-7 Small Regulatory RNA Is Controlled by Ecdysone during metamorphosis in Drosophila melanogaster. Sempere, L.F., Dubrovskaya, V.A., Dubrovsky, E.B., Berger, E.M., Ambros, V., Developmental Biology, vol. 244: 170-9 (2002).

Figure 2

Mass of Target Plasmid on Membrane

Figure 2. Dot Blot Assay Comparing Sensitivity of Kinase- and StarFire®-Labeled Probes. Serial dilutions of target plasmid pGreen Lantern-1 were spotted on duplicate nylon membranes at 100, 10, and 1amol. An oligonucleotide specific for pGreen Lantern-1 (#R514) was labeled using the PNK and StarFire methods. One filter strip was hybridized to the StarFire-labeled probe (top), while a duplicate was hybridized to the kinase-labeled probe (bottom). Hybridization, washes, and exposures were otherwise identical. [1]

Figure 3

Figure 3. Northern Blot Demonstration of pre-mRNA Splicing Intermediates in S. pombe. Northern blots were prepared using total RNA from S. pombe wild-type (wt) and strain prp2-1, a conditional lethal mutant that accumulates pre-mRNA splice products at 36°C but proceeds with splicing normally at 25°C. Duplicate blots were probed with ODN #667113, specific for intron 1 of the tf2d gene. One blot (left) was hybridized to ODN #667113 end-labeled with PNK, and a second blot (right) was hybridized to the same ODN labeled with the StarFire method. Hybridization conditions, washes, and exposures were otherwise identical. [1]