Guide for ordering of mircury LNA probes and LNA oligonucleotides

Transcription

1 1 Guide for ordering of mircury LNA probes and LNA oligonucleotides microrna mrna Other Detection, p.2-3 in situ hybridization, p.6-7 Other application, p.8-9 Kckdown (in vitro), p.4-5 Other mrna application, p.8-9 Other RNA target, p.8-9 Kckdown (in vivo), p.8-9 Other microrna application, p.8-9

2 mircury LNA Detection probes for microrna detection by ISH or Northern 2 Order XX mircury LNA Detection probe, 250 pmol for any microrna antated in Sanger mirbase. Select probe specific product # from Exiqon website Northern blot probes = in situ hybridization probes Pre-designed microrna probes full length based on mirbase Product number Product description XXXXXX-00 mircury LNA Detection probe, 250 pmol, ready to label U XXXXXX-01 mircury LNA Detection probe, 250 pmol, 5'-DIG labeled U XXXXXX-02 mircury LNA Detection probe, 250 pmol, 5'-ami labeled U XXXXXX-03 mircury LNA Detection probe, 250 pmol, 5'-biotin labeled U XXXXXX-04 mircury LNA Detection probe, 250 pmol, 5'-fluorescein labeled U XXXXXX-05 mircury LNA Detection probe, 250 pmol, 3'-DIG labeled U XXXXXX-06 mircury LNA Detection probe, 250 pmol, 3'-ami labeled U XXXXXX-07 mircury LNA Detection probe, 250 pmol, 3'-biotin labeled U XXXXXX-08 mircury LNA Detection probe, 250 pmol, 3'-fluorescein labeled U Positive/Negative controls Product No Product Name Product Description Sense mir-159 Control, mircury detection probe, 250 pmol, ready to label Scramble-miR Control, mircury detection probe, 250 pmol, ready to label U6, hsa/mmu/r Control, mircury detection probe, 250 pmol, ready to label Also available: custom designed microrna probes: If probe is t found or customer has special requests see next page for explanation to Exiqon custom probe products and which information to provide

3 Probe options for microrna detection by ISH or Northern 3 Probe for a microrna anted in Sanger mirbase or mirplus Standard Exiqon LNA detection probe (full length, different labels see p.2) Order from webshop: XX mircury LNA TM Detection probe, 250 pmol Need ather label If ather label is requested, request will be handled as custom order: Please send the following to tech support : - microrna name (e.g. dre-mir-16) as found in webshop Tech support will send ref# for order and confirm Exiqon list price Pricing and product specification follows custom mircury LNA TM Detection probe, 250 pmol + price of label Product will appear as a # Transfer price is to be calculated accordingly cf. contract Want standard detection probe designed by Exiqon (meaning: full lenght probe, customer has reservations) Need ather label Please send the following to tech support for probe design: - complete TARGET sequence (please specify) Tech support will make optimal LNA probe, send ref# for order Pricing and product specification follows: XX custom mircury LNA TM Detection probe, 250 pmol Please, clearly state that you send TARGET sequence, but for ather small RNA, e.g snrna, but with a mutation/mismatch/sense want standard detection probe designed by Exiqon (meaning: full lenght probe, customer has reservations) Please send the customer s probe design to tech support for approval as follows: - PROBE sequence - LNA spiking pattern (required) * - synthesis scale (200 or 1000 nmol, yield sent to customer is at least 10%)* - application if relevant Tech support will approve possibility of synthesis for customers design, send ref# and Exiqon list pricing information (incl. HPLC fee) for order. Distributor discount according to contract. *FOR MORE DETAILS on #500150, see p. 9 This request falls between two products (99999-XX and XX). Please send the following to tech support for probe design: - TARGET sequence (all of it) - name of RNA/desired probe name - animal model Tech support will make optimal LNA probe, inform what product type is available for request and send ref# for order. Design tips: 3 mirnas are expected to be nt. This is the optimal size for an LNA probe. Hence, all custom detection probes are designed as full length. 1. For microrna, we request to send us complete TARGET sequence. The more sequence, the better. This gives flexibility for design of the most optimal probe. 2. Most optimal use of LNA is obtained for probes at the length of nt. Probe sequences shorter than this are t recommended. For LNA probes longer than 30 nt, the Tm becomes too high or the effect of LNA will be diluted. 3. When possible we suggest to always supply TARGET sequence. In some cases it may be necessary to send PROBE sequence, e.g. if customer has a suggested mismatach probe. Please send the following to tech support for probe design: - PROBE or complete TARGET sequence (please specify) Tech support will make optimal LNA probe, send ref# for order Pricing and product specification follows: XX custom mircury LNA TM Detection probe, 250 pmol Please, clearly state whether you send TARGET or PROBE sequence

4 4 mircury LNA Kckdown probes for direct microrna inhibion Order XX mircury LNA Kckdown probe, 5 nmol for any microrna antated in Sanger mirbase. Select probe specific product # from Exiqon website Pre-designed micorna probes based on mirbase Product number Product description 1XXXXX-00 mircury LNA kckdown probe, 5 nmol, ready to label 1XXXXX-02 mircury LNA kckdown probe, 5 nmol, 5'-ami labeled 1XXXXX-04 mircury LNA kckdown probe, 5 nmol, 5'-fluorescein labeled 1XXXXX-06 mircury LNA Detection probe, 5 nmol, 3'-ami labeled 1XXXXX-08 mircury LNA Detection probe, 5 nmol, 3'-fluorescein labeled Positive/Negative controls Product No Product Name Product Description Sense mir-159 Control, mircury kckdown probe, 5 nmol, ready to label Scramble-miR Control, mircury kckdown probe, 5 nmol, ready to label Also available: custom designed microrna probes: If probe is t found or customer has special requests see next page for explanation to Exiqon custom probe products and which information to provide:

5 Probe options for microrna kckdown 5 Probe for a microrna anted in Sanger mirbase or mirplus Standard Exiqon LNA kckdown probe (different labels see p. 4) Order from webshop: XX mircury LNA TM Detection probe, 5 nmol Need ather label If ather label is requested, request will be handled as custom order: Please send the following to tech support : - microrna name (e.g. dre-mir-16) as found in webshop Tech support will send ref# for order and confirm Exiqon list price Pricing and product specification follows custom mircury LNA TM Kckdown probe, 5 nmol + price of label Product will appear as a # Transfer price is to be calculated accordingly cf. contract Want standard kckdown probe designed by Exiqon (meaning: customer has reservations) Need ather label Please send the following to tech support for probe design: - complete TARGET sequence (please specify) Tech support will make optimal LNA probe, send ref# for order Pricing and product specification follows: XX custom mircury LNA TM Kckdown probe, 5 nmol Please, clearly state that you send TARGET sequence, but for ather small RNA, e.g snrna, but with a mutation/mismatch/sense want standard kckdown probe designed by Exiqon (meaning:customer has reservations) Please send the customer s probe design to tech support for approval as follows: - PROBE sequence - LNA spiking pattern (required) - synthesis scale (200 or 1000 nmol, yield sent to customer is at least 10%)* - application if relevant Tech support will approve possibility of synthesis for customers design, send ref# and Exiqon end-user pricing information (incl. HPLC fee) for order. Distributor discount according to contract. Pricing and product specification follows: custom LNA oligonucleotides FOR MORE DETAILS see also p. 9 This request falls between two products ( XX and ) Please send the following to tech support for probe design: - TARGET sequence (all of it) - name of RNA/desired probe name - animal model Tech support will make optimal LNA probe, inform what product type is available for request and send ref# for order. Design tips (see also p. 9): mirnas are expected to be nt. This is the optimal size for an LNA probe. The most optimal design for a kckdown probe may t full length, but if Exiqon is to design: 1. For microrna (and other RNA), we request to send us complete TARGET sequence. The more sequence, the better. This gives flexibility for design of the most optimal probe. 2. Most optimal use of LNA is obtained for probes at the length of nt. Probe sequences shorter than this are t recommended. For LNA probes longer than 30 nt, the Tm becomes too high or the effect of LNA will be diluted. 3. When possible we suggest to always supply TARGET sequence. In some cases it may be necessary to send PROBE sequence, e.g. if customer has a suggested mismatach probe. Please send the following to tech support for probe design: - PROBE or complete TARGET sequence (please specify) Tech support will make optimal LNA probe, send ref# for order Pricing and product specification follows: XX custom mircury LNA TM Kckdown, 5 nmol Please, clearly state whether you send TARGET or PROBE sequence

6 mrna probe for in situ hybridization 6 Order XX mrna in situ probe, 5 nmol for custom designed probes for detection by ISH for any mrna target Standard product offering See next page for detailed explanation on how to order. Exiqon uses in-house software tools to design detection probe with optimal LNA spiking pattern. Product number Product name Product description Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, ready to label Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 5'-DIG labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 5'-ami labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 5'-biotin labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 5'-fluorescein labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 3'-DIG labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 3'-ami labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 3'-biotin labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 3'-fluorescein labeled Named by Customer LNA mrna in-situ hybridisation probe; 5 nmol, 5'-DIG and 3'-DIG labeled Positive/negative controls (standard label offering as above) Product number Product name Product description Scramble-ISH Control, LNA mrna in situ hybridisation probe, 5 nmol, ready to label PolyT(25)Vn Control, LNA mrna in situ hybridisation probe, 5 nmol, ready to label beta-actin, mmu/r Control, LNA mrna in situ hybridisation probe, 5 nmol, ready to label beta-actin, hsa Control, LNA mrna in situ hybridisation probe, 5 nmol, ready to label Also available (customer s own design or other label option): If desired probe:label combination is t found or customer has custom requests see next page for explanation to Exiqon custom mrna probe products and which information to provide:

7 mrna probe for in situ hybridization How to order Exiqon designed probe ( XX) or customers own design (500150) Customer wants Exiqon to design mrna probe for in situ hybridization 7 No, use own design or different application STANDARD CUSTOM PROBE EXIQON DESIGNS: XX mrna in-situ hybridization probe, 5 nmol (label options see p.6) Please send the following to tech support for probe design: - TARGET sequence (as much as possible t a reference or database. of actual nt sequence is required to avoid misunderstandings) - Organism (we BLAST the probe for potential risk of cross-hybridization) - Preferred label - Application Tech support will design optimal mrna probe (19-25 nt) with respect to LNA probe design criteria (see * below) and BLAST the probe against NCBI gene database. When probe is designed ref# will be sent to distributor for ordering. Pricing and product specification follows: XX mrna in-situ hybridization probe. LNA spiking pattern is t provided. Clearly confirm that TARGET sequence is forwarded CUSTOM CUSTOM PROBE Customer designs or customer has specific requirements: LNA TM Oligonucleotide (for a microrna target) Please send the following to tech support for probe design: - PROBE sequence with LNA spiking pattern, LNA shown as catipal letters, e.g. atgcatgca - synthesis scale (200 nmol or 1000 nmol yield sent to customer is at least 10%) - application (if relevant) Confirm that PROBE sequence is fowarded. (Probe is t blasted) Pricing and product specification follows: , see table for single DNA and LNA nucleotides. All probes are HPLC purified and will be added to pricing. See details on p. 9 Need ather label Pricing and product specification follows XX mrna in-situ hybridization probe + price of label. Product will appear as a # Transfer price is to be calculated accordingly cf. contract *Tips and information for LNA probe design: 1. The most optimal use of LNA is obtained for detection probes at the length of nt. This will ensure the most optimal LNA spike-in pattern and therefore the optimal Tm of the probe for specific binding and to avoid background. Shorter probes for detection are t recommended as they will be very difficult to design with Tm high eugh for stringent hybridization conditions and thus proper specific binding. For LNA probes longer than 30 nt, the Tm becomes too high or the effect of LNA will be diluted. 2. If Exiqon is to design: Please send as much TARGET sequence as possible - it will better ensure a customer satisfaction in the end. The more sequence, the better. This gives flexibility for designing the most optimal probe. We use our in-house software tools to design the most optimal detection probe in terms of LNA-spiking pattern, Tm, length (20-25 nt), GC content, minimal secondary structure and self-annealing properties. 3. It is recommended to avoid larger repeats of G in the probe. Longer repeats of G s as well as larger content of G will complicate the synthesis of the LNA probe resulting in lower yield or lower quality. 4. When possible we suggest to always supply TARGET sequence. In some cases it may be necessary to send PROBE sequence, e.g. if customer has a suggested mismatach probe. 5. LNA spiking pattern will only be provided to the customer if originally given by customer. LNA spiking pattern is t a prerequisite for publishing of data.

8 8 Custom LNA oligonucleotids (prod ) Yes, Exiqon can synthesize an LNA oligonucleotide of for customer s own design for any application Order per DNA/LNA base oligo at standard size synthesis 200 nmol (customer receives min. 20 nmol) or large size synthesis 1000 nmol (customer receives min. 100 nmol). See next page for details. Type Nomenclature LNA A,G,mC,T DNA a,c,g,t 5 -Biotin (C4- C6) Bio 3 -Biotin (C4- C6) Bio 5 -Ami NH2C6 3 -Ami NH2C6 5 -FAM Fitc 3 -Quencher2 #Q2 5 -DIG* Dig 3 -DIG* Dig 5 -Cy3 Cy3 5 -Cy5 Cy5 HPLC - NB: For microrna or mrna detection probes and microrna kckdown probes, we recommend that Exiqon design the probe for optimal LNA content and positioning. See previous pages for products: XX, XX, and XX, respectively. For in vivo kckdown probes please contact Tech support at support@exiqon.com

9 LNA TM Oligonucleotides Ordering of product custom custom probes 9 High quality LNA Oligonucleotides are available for a variety of different specialty applications and invative products. LNA applications: microrna analysis in situ hybridization (detection) Northern blotting (detection) Kckdown capture probe design for microarrays primers for qpcr In vivo kckdown Gene expression analysis by real-time PCR Probes for cytogenetics (FISH) Probes for mrna in situ hybridisation mrna isolation microrna antisense molecules SNP detection mrna antisense molecules Allele-specific PCR priming sirna DNAzymes Fluorescence Polarization probes Molecular Beacons Capture probes for expression microarrays Comparative geme hybridisation (CGH) LNA provides superior binding and hybridization characateristics as well as enhanced biostability. LNA oligonucleotides can be used with great advantage in any application where high hybridization specificity is needed, and in particular when the target sequence is relatively short. The high binding affinity of LNA allows for the use of short and very specific probes LNA TM Oligonucleotide Customer designs or has specific requirements: When: A. For probes fully designed by the customers themselves What is offered:* - Custom oligos priced per nt base - HPLC always included (remember to include this in pricing to customer!) - Desalting t possible/t as standard nmol or 1000 nmole SYNTHESIS scale (yield sent to customer is at least 10%)** - probe sent in solution. Can be dried down upon special request. Procedure for ordering: 1. Send probe information to Exiqon tech support (support@exiqon.com) - PROBE sequence with LNA spiking pattern, LNA shown as catipal letters, e.g. atgcatgca - synthesis scale (200 nmol or 1000 nmol yield sent to customer is at least 10%)* - application (if relevant or possible) 2. Exiqon Tech support will design probe and confirm Exiqon end-user pricing (incl. HPLC fee) with a ref #. Please use ref # for ordering. IMPORTANT: customer designed LNA oligos are more difficult to synthesize. Some designs may t be possible to make, therefore a confirmation (i.e. ref #) from Exiqon is always required when placing the order. 3. Place order with ref# 4. Exiqon will synthesize the probe (shipment after 10 working days) Tips for customer design: 1. The most optimal use of LNA is obtained for probes at the length of nt. This will allow and ensure the most optimal combination of LNA spike-in pattern and Tm of the probe for specific binding and to avoid background. Typically the LNA content should be more than 25% but t exceed 50%. 1. Shorter probes for detection are t recommended as they will be very difficult to design with Tm high eugh for proper specific binding. 2. Longer LNA probes >30 nt are difficult to synthesize. For LNA probes longer than 30 nt, the Tm becomes too high (as too many LNA nt). Lower Tm would be obtained with fewer LNA nt, but then the effect of LNA will be diluted. 2. Refer customer to Tm tool on Exiqon website for help in their design of LNA oligo probe 3. It is recommended to avoid larger repeats of G. Longer repeats of G s as well as larger content of G will complicate the synthesis of the LNA probe resulting in lower yield or lower quality. 4. LNA spiking pattern will only be provided to the customer if originally given by customer. LNA spiking pattern is t a prerequisite for publishing of data. 9 ** yield depends on the ease of the synthesis. Many LNAs, long repetition of G s, and secondary structure will provide lower yield. Yield is typical between 15-20% NB: For microrna or mrna detection probes and microrna kckdown probes, we recommend that Exiqon design the probe for optimal LNA content and positioning. See previous pages for products: XX, XX, and XX respectively.