For Fluorescent Differential Display with three fluorescently labeled one-base anchored oligo-dT primers and rationally designed arbitrary 13mers. The RNAspectra Kits feature a new generation of Differential Display technology based on fluorescent detection of PCR products instead of radioactivity. It is the first non-radioactive differential display system with similiar sensitivity to that of 33P isotopic labeling. Each kit contains all the reagents needed for fluorescent differential display except RNA samples, Taq DNA polymerase, and a fluorescent scanner. The MessageClean® is also highly recommended prior to differential display to remove trace amounts of DNA contamination from RNA samples. GenHunter currently sells two versions of the RNAspectra Kit: a "Green" and a "Red" kit. As in the RNAimage® Kit, the RNAspectra kit contains three one-base-anchored oligo-dT primers to subdivide the mRNA population, except they are labeled with either 5' Fluorescein (RNAspectra Green Kits) or 5' Rhodamine (RNAspectra Red Kits). The use of three one-base-anchored primers reduces the redundancy and potential under-representation of the sub-population of mRNAs encountered by using either twelve individual or four degenerate two-base-anchored oligo-dT primers. With built-in restriction sites at the 5' ends of both anchored and arbitrary 13mers, the longer primer pairs produce highly selective and reproducible cDNA patterns. These improvements ensure that differentially expressed genes are more readily identified, cloned, and manipulated. See Technical Notes Section for further discussion on primer design, calculation of coverage, comparison of DD to other methodologies, and false positives. Comparison of Fluorescent DD (FDD) and isotopic DD. RNA samples from normal (N) and tumor (T) specimens were compared using isotopic DD and FDD. Each image uses a one-base anchored oligo-dT primer, H-T11G, in duplicate with one arbitrary primer. Note the same differences are seen and the excellent reproducibility. Fluorescent DD Image using the RNAspectra Red Kit Comparison of 4 RNA samples (before and 6, 9, and 12 hours after a drug treatment) with automated reaction setup. Digital FDD spectra analysis Digital quantatative analysis of an FDD gel spectra accurately reveals a drug inducible gene (blue arrow). The red and green spectra represent different RNA samples being compared. Schematic representation of Fluorescent mRNA Differential Display using RNAspectra Kits. Three one-base anchored oligo-dT primers with 5' HindIII sites are used in combination with a series of arbitrary 13mers (also containing 5' HindIII sites) to reverse transcribe and amplify the mRNAs from a cell. Fluorescently labeled anchor primers are used in PCR. See Technical Notes for more detailed information on the optimal primer designs for differential display.   Cat. No. RNAspectra Green Kit 1 F501 RNAspectra Green Kit 2 F502 RNAspectra Green Kit 3 F503 RNAspectra Green Kit 4 F504 RNAspectra Green Kit 5 F505 RNAspectra Green Kit 6 F506 RNAspectra Green Kit 7 F507 RNAspectra Green Kit 8 F508 RNAspectra Green Kit 9 F509 RNAspectra Green Kit 10 F510 RNAspectra Red Kit 1 R501 RNAspectra Red Kit 2 R502 RNAspectra Red Kit 3 R503 RNAspectra Red Kit 4 R504 RNAspectra Red Kit 5 R505 RNAspectra Red Kit 6 R506 RNAspectra Red Kit 7 R507 RNAspectra Red Kit 8 R508 RNAspectra Red Kit 9 R509 RNAspectra Red Kit 10 R510 RNAspectra Kits 2-10 contain all the components of Kit 1, but each has 8 different arbitrary 13mers (H-AP9 to H-AP80). Choosing Kits: One could start with any RNAspectra Kit since the sequences are random and designed to be maximally different from one another, but to ensure a maximum coverage, multiple kits may be needed (See Technical Notes). The RNAspectra Kits have been optimized for use with the Hitachi Genetic Systems FMBIO® II or III Fluorescent Imaging Systems. Contact GenHunter for more information on the FMBIO® II or III.". Individual components for the RNAspectra Kits sold separately Description Cat. No. Volume FH-T11G Green Anchor Priner F101-G 200 µL FH-T11C Green Anchor Priner F101-C 200 µL FH-T11A Green Anchor Priner F101-A 200 µL FH-T11N Green Anchor Priner Set F101-S 200 µL each RH-T11G Red Anchor Priner R201-G 200 µL RH-T11C Red Anchor Priner R201-C 200 µL RH-T11A Red Anchor Priner R201-A 200 µL RH-T11N Red Anchor Priner Set R201-S 200 µL each H-AP Arbitrary Primers (2 µM each) H-AP1 to H-AP80 150 µL H-AP Primer Set 1 (H-AP1 to H-AP8) H-AP-A 150 µL H-AP Primer Set 2 (H-AP9 to H-AP16) H-AP-B 150 µL H-AP Primer Set 3 (H-AP17 to H-AP24) H-AP-C 150 µL H-AP Primer Set 4 (H-AP25 to H-AP32) H-AP-D 150 µL H-AP Primer Set 5 (H-AP33 to H-AP40) H-AP-E 150 µL H-AP Primer Set 6 (H-AP41 to H-AP48) H-AP-F 150 µL H-AP Primer Set 7 (H-AP49 to H-AP56) H-AP-G 150 µL H-AP Primer Set 8 (H-AP57 to H-AP64) H-AP-H 150 µL H-AP Primer Set 9 (H-AP65 to H-AP72) H-AP-I 150 µL H-AP Primer Set 10 (H-AP73 to H-AP80) H-AP-J 150 µL dNTP Mix (FDD) S505 500 µL 10X PCR Buffer S201 500 µL 5X RT Buffer S401 500 µL MMLV Reverse Transcriptase (100 units/µL) S402 40 µL FDD Loading Dye F201 1 mL H-T11G Anchor Primer (2µM) H-101-G 300 µL H-T11A Anchor Primer (2µM) H-101-A 300 µL H-T11C Anchor Primer (2µM) H-101-C 300 µL H-T11N Anchor Primer Set (2µM each) H-101-S 300 µL ea. dH2O DH2O 1.2 mL Fluorescein Locator Dye F202 100 µL

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