FAQs of Fluorescent RNA

Frequently Asked Questions

 

1. Q: How do I start to apply Pepper Technology to my research?

A:Pepper reagents are consisting of both the HBC dye, and plasmids encoded Pepper RNA.  Customers should include a positive control plasmids (PET2808MCS0 for E. coli or  PAPU608MCS0 for mammalian cells)  in their first order along with their choice of HBC dyes; alternatively, customers may order the Pepper Fluorescent RNA  starter kits (Cat#  PET2808KIT or  PAPU608KIT).  The copy numbers of RNAs are usually 3-4 orders less than proteins, therefore imaging of RNAs in live cells remains to be challenging. Although Peppers have much improved brightness and signal-noise ratio comparing to other live cell labeling technologies, experiments using Pepper labeling should be done with caution and well controlled. For starters, we strongly suggest that you work first with vectors expressing Pepper or Pepper tandem arrays as positive control, before test your own constructs encoding target RNAs. Test the transfection, staining and imaging of these Pepper or Pepper tandem arrays as positive control in 293T cells, Cos7 cells or Hela cells. Then test these positive controls in your own cells. You may optimize the conditions or parameters for cell culture, transfection, staining or imaging in order to obtain the best Signal/Background ratio. Then you may proceed with your target RNA labeled with Pepper. 

 

2. Q: What kind of instruments are compatible with Pepper technology

A: Pepper fluorescent RNA may be detected by many general instruments that capable of detect fluorescent signals, such as microscope, flow cytometer, microplate reader or Quantitative PCR cycler.

 

3. Q: Can I observe live cells expressing Pepper fluorescent RNAs with naked eye?

A: Yes, you can easily observe cells expressing Pepper fluorescent RNAs with naked eye using a fluorescent microscope.

 

4. Q: Which HBC ligand is the best?

A: There are 8 HBC ligands of different colors. It is your call to choose the right HBC ligand for your application, which may be determined by the nature of your cells, applications and the set up of your instrument. Keep in mind that colors, brightness, photostability and Signal/Background ratio are important factors to obtain good results. We suggest you to start with the HBC Ligands kit (8 color in total) and test the performance of each HBC ligands on your cells and your instruments. Each instrument has different excitation light source and excitation/emission filters, therefore you may test and choose the HBC dyes that are best compatible with your instrument. Different HBC ligands may have different background signal in certain cells and this must be tested. In general, all the ligands works well in E. coli cells, while HBC485\ HBC497\ HBC514\ HBC530\ HBC620 ligands have adequate brightness and low background in human cells. Among them, Pepper complexed with HBC497 and HBC530 has the best Signal/Background ratio and are recommended for the detection of RNAs of low copy numbers, however they photoswitch reversibly under strong excitation light, therefore they are not suitable for fast confocal imaging (fast two photon imaging, or slow confocal imaging at 1 frame/min would be OK) or SIM imaging. Pepper complexed with HBC485 and HBC620 are quite photostable, and are recommended for fast confocal imaging and SIM imaging.

 

5. Q:Which size of the HBC product should I choose?

A: Please use the following table to determing the size of the HBC dyes. Please be noted that the amount of each HBC dyes may be different for the same product size, however, the volume of stock solution and the sample numbers remain the same.

 

Size of HBCs	Stock solution (ul)	Working solution (ml)	Sample numbers (96 well plate)	Sample numbers  (35 mm dish)
T	30	30	600	60
S	60	60	1200	120
L	200	200	4000	400

* Minimal volume of working solution for different sample formats: 5 ul for staining samples on cover glass; 50 ul per well for 96 well microplate; 0.5 ml per 35 mm imaging dish. For best results, higher volume of working solution may be also used.

 

6. Q:What is the storage and transport conditions for HBC ligands?

HBC dyes are stable at ambient temperture for several months, therefore they are shipped under ambient tempeature without significant effects on their quality, integrity, and functional performance. For long term storage, HBC dyes may be stored at -20°C and remain active for at least 18 months. Reconstitute the HBC dyes with anhydrous DMSO according to the label on the vial (30 ul for T size, 60 ul for S size, or 200 ul for L size) to make 1000X stock solutions. Store the solution at -20°C, and protect them from light. The stock solution may be dispensed into aliquots for storage, to avoid multiple freeze-thaw cycles. The stock solution remains active for at least three months.

 

7. Q:Have you test Pepper RNA in live animals?

A: No.

 

8. Q: Can I pay through bank wire transfer?

A: Yes. Please contact us through email, indicate which products to be ordered. We will send you an invoice for the bank wire transfer. 

 

9. Q: Can I pay with credit cards?

A: Not currently.

 

10. Q: Can I pay through Paypal?

A: Yes. Customers outside China may pay with Paypal express checkout.

 

11. Q: What about shipping after the purchase?

A: We ship through Fedex or International Express Mail. Once the payment is confirmed, we will arrange the shipment and email you the Tracking#.

 

12. Q: Does your price quote include tax for customers outside China?

A: The price quote does not include any tax for customers outside China. The customers are solely responsible for any taxes or duties that may incurred for our product in your country.