Frequently Asked Questions | Nucleic Acid Labeling

Label IT® Nucleic Acid Labeling Kits

The Label IT® Nucleic Acid Labeling Kits offer efficient, one-step, direct labeling for any type of nucleic acid. This non-enzymatic labeling method covalently attaches labels to nucleic acids in a non-destructive manner. The Label IT® Kits are available with a variety of label options including Cy®3, Cy®5, CX-Rhodamine, TM-Rhodamine, Fluorescein, MFP488, Digoxin, Biotin, and Dinitrophenyl (DNP). Label IT® Nucleic Acid Modifying Kit, Amine is also available for the modification of DNA or RNA with amine functional groups. In addition to the general Label IT® Nucleic Acid Labeling Kits, optimized labeling kits are available for the following applications:

• In vitro and in vivo tracking of plasmid DNA (Label IT® Tracker™ Kits)
• In vitro and in vivo tracking of siRNA (Label IT® siRNA Tracker™ Kits)

The general Label IT® Kits are featured in this FAQ. For questions specific for the other kits, please visit the respective web pages or contact Technical Support.

GENERAL QUESTIONS AND ANSWERS

Q1. How do the Label IT® Reagents label nucleic acids?
Q2. What types and sizes of nucleic acids can I label with the Label IT® Reagents?
Q3. What labeling efficiencies can I expect if I follow the Label IT® Reagent protocol?
Q4. Can I label dNTPs with the Label IT® Reagents?
Q5. Can proteins be labeled?
Q6. Does labeling using Label IT® Reagents affect nucleic acid functionality?

PROTOCOL QUESTIONS AND ANSWERS

Q7. How should I store the Label IT® Reagents and the labeled nucleic acid?
Q8. Can I scale the labeling reaction up or down?
Q9. How will increased incubation times affect the labeling efficiency?
Q10. How will increasing the amount of Label IT® Reagent (per 1 ug nucleic acid) affect the labeling efficiency?
Q11. Once I have labeled my sample, how can I avoid cross-labeling any other DNA that I subsequently add to my sample?
Q12. How do I know if my nucleic acid is labeled?
Q13. What is the recommended protocol for estimating the number of fluorescent labels per nucleic acid molecule?
Q14. Can I directly visualize nucleic acids labeled with Digoxin, DNP or Biotin?
Q15. Can I directly visualize nucleic acids labeled with CX-Rhodamine, TM-Rhodamine, Fluorescein, MFP488, Cy®3,or Cy®5 on a gel?
Q16. What are the excitation and emission wavelengths for CX-Rhodamine, TM-Rhodamine, Fluorescein, MFP488, Cy®3 and Cy®5 dyes?
Q17. Can the quality and concentration of nucleic acid affect the labeling reaction?
Q18. Why should labeled DNA probes be denatured with the Denaturation Reagent D1?
Q19. Can labeled RNA be denatured with the Denaturation Reagent D1?
Q20. Will dithiothreitol (DTT) or RNase inhibitors have an effect on the labeling reaction?

Label IT APPLICATIONS

Q21. Can labeled DNA be transfected?
Q22. Can labeled siRNA be transfected?
Q23. Can labeled DNA be used for DNA-binding studies such as gel-shift assays?
Q24. Can I use the Label IT® Kits for traditional hybridization applications?
Q25. Can I use the Label IT® Reagents for FISH applications?
Q26. Can I use the Label IT® Reagents for microarray applications?
Q27. Can I use the Label IT® Reagents to attach my DNA to a glass slide or bead?

GENERAL QUESTIONS AND ANSWERS

Q1. How do the Label IT® Reagents label nucleic acids?
The Label IT® Labeling Reagent comprises a reactive alkylating agent with strong nucleic acid binding capability facilitated via electrostatic interactions. Labeling via covalent modification (alkylation) of RNA or DNA can take place on reactive heteroatoms on any nucleotide of the nucleic acid polymer.

Q2. What types and sizes of nucleic acids can I label with the Label IT® Reagents?
Any purified nucleic acid can be labeled using the Label IT® Reagents, e.g., plasmid DNA, siRNA, oligonucleotides, etc. We routinely label nucleic acids as short as 20 nucleotides and as long as many kilobases. We recommend using oligonucleotides greater than 5 bases in length to ensure efficient labeling.

Q3. What labeling efficiencies can I expect if I follow the Label IT® Reagent protocol?
Using the general Label IT® Nucleic Acid Labeling Kits, we routinely achieve one label every 20-60 base pairs estimated with a spectrophotometric assay for the fluorescent Label IT® dyes and DNP. See Q13 for details about calculating the labeling efficiency of your sample. Labeling densities are optimized for each particular application-specific labeling kit.

Q4. Can I label dNTPs with the Label IT® Reagents?
We do not recommend the use of the Label IT® Reagents to modify dNTPs. We recommend using oligonucleotides greater than 5 bases in length to ensure efficient labeling.

Q5. Can proteins be labeled?
Yes. The Label IT® Reagents can label primary amines that are present on proteins. However, the Label IT® Reagents have a higher affinity for nucleic acids than for proteins and therefore label proteins rather inefficiently. On the other hand, if there is a high concentration of protein or Label IT® Reagent in the reaction, proteins can be labeled.

Q6. Does labeling using Label IT® Reagents affect nucleic acid functionality?
Labeling density needs to be carefully optimized for each specific application. We have optimized the labeling densities for several different applications such that it does not interfere with the functional assay, as follows:

PROTOCOL QUESTIONS AND ANSWERS

Q7. How should I store the Label IT® Reagents and the labeled nucleic acid?
Store dried and reconstituted Label IT® Reagents and labeled nucleic acids tightly capped at -20°C. Protect the Label IT® Reagents from exposure to light and moisture. Improper storage may result in decreased labeling efficiencies. Fluorescently labeled nucleic acids must also be stored protected from light.

Q8. Can I scale the labeling reaction up or down?
Yes. It is possible to change the labeling density, the volume of the reaction and the amount of nucleic acid that is labeled. To modify the labeling density, simply change the ratio of the labeling reagent to nucleic acid during the labeling reaction or adjust the incubation time of the labeling reaction. In addition, the volume of the labeling reaction may be scaled up or down, simply use an amount of Label IT® Reagent that does not consist of more than 20% of the total reaction volume and the Labeling Buffer A diluted to 1X final concentration in the reaction. Refer to Tips from the Bench – Adjust Labeling Density Using Label IT® Reagents for further details.

Q9. How will increased incubation times affect the labeling efficiency?
When using the general Label IT® Nucleic Acid Labeling Kits, we observe a linear increase in labeling efficiency during the first 3 hours of incubation at 37°C. We recommend a standard reaction for 1 hour at 37°C. However, due to the linear nature of the labeling reaction, you can use twice as much Label IT® Reagent and incubate for 30 minutes at 37°C and achieve similar labeling efficiency.

Q10. How will increasing the amount of Label IT® Reagent (per 1 ug nucleic acid) affect the labeling efficiency?
Generally, modest changes (2-4 fold) in the ratio of Label IT® Reagent to nucleic acid affect the labeling efficiency in a linear manner. We do not recommend greater than 4-fold increase to the 1:1 (μl Label IT® Reagent : μg DNA) ratio; this may increase the chance of nicking the nucleic acid template. When scaling the labeling reaction, the amount of Label IT® Reagent should never constitute more than 20% of the total reaction volume.

Q11. Once I have labeled my sample, how can I avoid cross-labeling any other DNA that I subsequently add to my sample?
The covalent bonds between the label and the nucleic acids bases are very stable and will not allow cross labeling or transfer of labels to other species.

Q12. How do I know if my nucleic acid is labeled?
Depending upon the nature of the Label IT® dye used – fluorescent or epitope based, several methods can be used to ascertain whether your labeling reaction has been successful. For details, please see our Tips from the Bench – Determine Labeling Success Using Label IT® Reagents.

Q13. What is the recommended protocol for estimating the number of fluorescent labels per nucleic acid molecule?
The most straight-forward way to estimate the number of fluorescent labels on the DNA (or RNA) molecule involves measuring the absorbance of the nucleic-dye conjugate at 260 nm (A₂₆₀) and the λMAX for the particular dye (A_dye). To quantify your labeled sample, you can either calculate the base: Label IT® dye ratio or pmol of dye per μg of nucleic acid. For details on these quantification methods, please see Tips from the Bench – Calculate the Labeling Density of your Sample.

Q14. Can I directly visualize nucleic acids labeled with Digoxin, DNP or Biotin?
Yes. On an ethidium bromide-stained agarose gel, you may see a slower migration rate with labeled DNA compared to unlabeled control DNA. Alternatively, secondary detection can be performed using suitable antibody or avidin conjugates and substrates for dot blot applications.

Q15. Can I directly visualize nucleic acids labeled with CX-Rhodamine, TM-Rhodamine, Fluorescein, MFP488, Cy®3,or Cy®5 on a gel?
Yes. Fluorescently labeled nucleic acid can be analyzed by agarose gel electrophoresis (without ethidium bromide staining) and then visualized on a UV transilluminator. The bands will appear faint because UV bulbs in the transilluminator emit light at approximately 300 nm (for a standard transilluminator with “midrange” bulbs). This is not the optimal excitation wavelength for the fluorescent labels.

Q16. What are the excitation and emission wavelengths for CX-Rhodamine, TM-Rhodamine, Fluorescein, MFP488, Cy®3 and Cy®5 dyes?
Following are the optimum excitation and emission wavelengths for the indicated Label IT® fluorophores.

Q17. Can the quality and concentration of nucleic acid affect the labeling reaction?
Yes. For best results, use purified nucleic acid in labeling reactions. For most applications, it is ideal to use concentrated stocks (~1 mg/ml) of the nucleic acid.

Q18. Why should labeled DNA probes be denatured with the Denaturation Reagent D1?
This alkaline denaturation process stabilizes the labeled DNA product and has shown increased hybridization sensitivity when compared to heat denaturation alone. If necessary, heat denaturation can be performed after an initial D1/N1 treatment.

Q19. Can labeled RNA be denatured with the Denaturation Reagent D1?
No. Alkaline conditions destroy RNA. RNA should be denatured by heating at 65°C for 10 minutes.

Q20. Will dithiothreitol (DTT) or RNase inhibitors have an effect on the labeling reaction?
DTT will have no effect on the labeling reaction, but proteinacious RNase inhibitors that are added before the labeling reaction will compete modestly with the nucleic acid. If the RNase inhibitors are added after the labeling is performed, they will have no effect on the product.

Label IT APPLICATIONS

Q21. Can labeled DNA be transfected?
Yes. We have successfully delivered labeled DNA into a variety of cell types using TransIT® transfection reagents for DNA transport and gene expression studies. Highly labeled nucleic acid may inhibit transcription. For gene expression studies, we recommend Label IT® Tracker™ Intracellular Nucleic Acid Localization Kits that provide an optimized labeling density for such applications.

Q22. Can labeled siRNA be transfected?
Yes. We have successfully delivered labeled siRNA into a variety of cell types using our TransIT-X2®, TransIT-TKO® and TransIT-siQUEST® Transfection Reagents for siRNA tracking and gene suppression studies. Our Label IT® siRNA Tracker™ Kits were developed for this purpose and do not interfere with RNAi knockdown.

Q23. Can labeled DNA be used for DNA-binding studies such as gel-shift assays?
Since the Label IT® Reagent covalently modifies nucleic acid residues, it may interfere with the binding of some proteins. The ratio of Label IT® Reagent to DNA can be modified during the labeling reaction to allow for fewer labeled residues per DNA molecule; however, the DNA will have to be tested in each specific binding application to determine biological activity.

Q24. Can I use the Label IT® Kits for traditional hybridization applications?
Yes. The Label IT® Reagents can be used for the preparation of non-radioactive probes for hybridization applications. Labeled DNA probes can be easily substituted in your standard hybridization and detection protocol.

Q25. Can I use the Label IT® Reagents for FISH applications?
The Label IT® Technology is ideal for the preparation of probes for FISH (probing of specific DNA sequences on chromosomes in metaphase spreads and/or interphase nuclei) applications. Depending on the quantity of repetitive sequences in the chosen FISH probe, the addition of blocking DNA may be necessary to reduce non-specific background signal. The most common blocking DNAs are species-specific cot-1 DNA and sheared salmon sperm DNA.

Q26. Can I use the Label IT® Reagents for microarray applications?
The Label IT® Nucleic Acid Labeling Kits can be optimized for labeling microarray samples such as mRNA, cDNA, cRNA and miRNA. Additional detection reagents (not supplied) may be required for microarray analysis. Other sample types (e.g. ribosomal RNA, bacterial RNA, genomic DNA) can also be labeled with the Label IT® Labeling Kits.

Q27. Can I use the Label IT® Reagents to attach my DNA to a glass slide or bead?
The Label IT® Modifying Reagent Kit directly modifies nucleic acids with amine functional groups and is designed for custom dye or solid surface attachment chemistry. For example, amine modified nucleic acids can be attached to silylated (aldehyde) slides or beads.