Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation

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Dec 2018



Northern blot is a molecular biology technique that can detect, quantify, and determine the molecular weight of RNA. Recently, we published a protocol utilizing near-infrared (IR) fluorescent probes in Northern blot (irNorthern). Our method is as sensitive as other non-radioactive methods but is more straightforward and versatile. Additionally, we found that IR-labeled probes can be used to multiplex or detect different species of RNA at the same time. Here we describe three methods for generating an IR-labeled probe as well as how to perform irNorthern blot. In conclusion, our irNorthern protocol offers a convenient method for RNA detection.

Keywords: Northern blot (Northern杂交), Near infrared (近红外), irNorthern (irNorthern), RNA detection (RNA检测), Non-radioactive (非放射性的)


Northern blot analysis is one of the foundational techniques available to biochemists and molecular biologists. While newer and more high-throughput techniques have been adopted, the Northern blot remains a powerful and adaptable method of analyzing RNA size and quantity simultaneously. In brief, DNA or RNA oligonucleotides labeled with radioactive phosphorus-32 (32P) are hybridized to a membrane crosslinked with RNA and subsequently detected using autoradiography (Alwine et al., 1977). Nonetheless, the use of 32P is subject to institutional regulation, and safety precautions must be observed during its use (Jones, 2005). Additionally, 32P probes have short lifespans increasing their cost for use. Alternative protocols using digoxigenin (DIG) conjugated to dNTP or NTP have been developed to replace 32P (Seibl et al., 1990). While successful, DIG probes require secondary detection by antibodies, more hands-on time and expensive reagents, deterring its wider adoption. Our recently published method, irNorthern, is straightforward and adaptable. It utilizes IR dyes which reduce auto-fluorescent background (Zarnegar et al., 2016; Miller et al., 2018). These IR dyes are conjugated with a dibenzocyclooctyne group (DBCO), which forms a covalent bond with the azide on the probes through copper-free click chemistry. To demonstrate its applications, we performed irNorthern to detect microRNA let-7a and small nuclear RNA U6 in total RNAs extracted from HCT116 wild-type and Drosha knockout cells (Kim et al., 2016). In addition, we provide details on how to generate three different azide-containing DNA or RNA probes for IR-dye labeling: a chemically synthesized DNA probe with an internal azide modification on dT, a DNA probe labeled with azide-dU by terminal transferase (TdT), and an RNA probe labeled with azide-U by T7 RNA polymerase.

Materials and Reagents

  1. Seal-Rite 1.5 ml microcentrifuge tube (USA Scientific, catalog number: 1615-5599)
  2. Hybridization tube (Fisher Scientific, catalog number: 13-247-300)
  3. Hybond N+ 30 cm x 3 M Roll (GE, catalog number: RPN303B)
  4. 50 ml centrifuge tubes (Genesee, catalog number: 21-106)
  5. 15 ml centrifuge tubes (Genesee, catalog number: 21-101)
  6. 1,250 µl pipet tips (USA Scientific, catalog number: 1112-1720)
  7. 0.1-10/20 µl pipet tips (USA Scientific, catalog number: 1110-3700)
  8. 200 µl pipet tips (USA Scientific, catalog number: 1110-1700)
  9. Whatman Grade 3 MM Chr blotting paper, sheet, 46 x 57 cm (GE, catalog number: 3030-917)
  10. 6-Well cell culture plates (Genesee Scientific, catalog number: 25-105)
  11. Paper towels (Highmark, catalog number 4497A1)
  12. Glass tray (Pyrex, catalog number: 1124847)
  13. Aluminum foil roll (Fisher, catalog number: 01-213-102)
  14. Sheet protectors (Office Depot, catalog number: 491694)
  15. HCT116 Parental cell line (Korean Collection for Type Cultures, catalog number: BP1230983)
  16. HCT116 Drosha KO #40 cell line (Korean Collection for Type Cultures, catalog number: BP1230984)
  17. let-7a irNorthern probe (5′-AACTATACAACCTACTACCTCA/iAzideN/A-3′)
    1. Probes are ordered from Integrated DNA Technology at 100 nmole scale, with one internal azide, and purified by HPLC.
    2. DNA probes are designed to be complementary to the target RNA. For miRNA targets, the probe is the full length (~22 nts) reverse complement of the mature miRNA. An internal azide is added close to the 3′ end with a terminal nucleotide as required for the synthesis.
  18. U6 irNorthern probe (5′-GCAGGGGCCATGCTAATCTTCTCTGTATCG/iAzideN/T-3′)
    Note: Probe sequences for commonly used target RNAs can be found in the literature. When designing new DNA probes, we recommend use ~30 nt sequence targeting single-stranded regions of the RNA.
  19. Terminal transferase (NEB, catalog number: M0315)
  20. 5-Azidomethyl-dUTP (Jena Biosciences, catalog number: CLK-084S)
  21. 5-Azido-C3-UTP (Jena Biosciences, catalog number: NU-157S)
  22. Agencourt AMPure XP beads (Beckman Coulter, catalog number: A63881)
  23. Isopropanol (J.T. Baker, catalog number: 908401)
  24. Ethanol (Decon Laboratories, catalog number: 2716)
  25. IRDye 680RD DBCO Infrared Dye, 0.5 mg (Li-Cor, catalog number: 929-50005)–light sensitive
  26. IRDye 800CW DBCO Infrared Dye, 5 mg (Li-Cor, catalog number: 929-55000)–light sensitive
  27. Urea (Sigma, catalog number: U5378)
  28.  Acryl/Bis 19:1 (VWR, catalog number: 97064-990)
  29. Ammonium persulfate (APS) (AmericanBio, catalog number: AB00112)
  30. TEMED (N,N,N',N'-Tetramethylethylenediamine) (AmericanBio, catalog number: AB02020)
  31. Sodium chloride (AmericanBio, catalog number: AB01915)
  32. Sodium phosphate dibasic anhydrous (Fisher Scientific, catalog number: BP332-500)
  33. Potassium chloride (Sigma, catalog number: P9541)
  34. Sodium acetate (Sigma, catalog number: S7545)
  35. Trizma base (Sigma, catalog number: T1503)
  36. HEPES (Sigma, catalog number: H4034)
  37. Ethylenediaminetetraacetic acid (EDTA) (Sigma, catalog number: EDS)
  38. Sodium Dodecyl Sulfate (SDS) (AmericanBio, catalog number: AB01920)
  39. Formamide DI (AmericanBio, catalog number: AB00600-00500)
  40. Bromophenol blue (Sigma, catalog number: 114391)
  41. Xylene Cyanol FF (Sigma, catalog number: X4126)
  42. Sodium Citrate Dihydrate (Sigma, catalog number: W302600)
  43. Phenol/Chloroform/Isoamyl Alcohol (Fisher Scientific, catalog number: BP1752I-400)
  44. Chloroform (AmericanBio, catalog number: AB00350-01000)
  45. ExpressHyb hybridization solution (Clontech, catalog number: 636832)
  46. TRIzol (Ambion, catalog number: 15596018)
  47. SYBR Green I Nucleic Acid Gel Stain (Invitrogen, catalog number: S7563)
  48. A, C, U, G ribonucleotides (Thermo Fisher Scientific, catalog number: R0481)
  49. Spermidine trihydrochloride (Sigma, catalog number: 85578)
  50. RNase Inhibitor, Murine (NEB, catalog number: M0314L)
  51. Dithiothreitol (DTT) (AmericanBio, catalog number: AB00490-00025)
  52. Magnesium chloride hexahydrate (Sigma, catalog number: M2670)
  53. HYCLN McCoys 5A Media (Fisher Scientific, catalog number: SH30200)
  54. Fetal bovine serum (PAA Laboratories, catalog number: A15-201)
  55. Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15070063)
  56. RQ1 RNase-Free DNase (Promega, catalog number: M6101)
  57. 1x Phosphate Buffered Saline (PBS) (see Recipes)
  58. RNA Elution Buffer (see Recipes)
  59. 8 M urea 15% polyacrylamide gel (see Recipes)
  60. 8 M urea 6% polyacrylamide gel (see Recipes)
  61. 2x Formamide Loading Dye (see Recipes)


  1. 16-Tube SureBeads magnetic rack (Bio-Rad, catalog number: 1614916)
  2. Vortex Genie 2 (Fisher Scientific, catalog number: 12-812)
  3. Micropipettes (Eppendorf, Research Plus Models)
  4. Roto-Bot rotating mixer (Benchmark Scientific, catalog number: R4045)
  5. Water-Jacketed CO2 incubator (Forma Scientific, catalog number: 3110)
  6. ThermoMixer C (Eppendorf, catalog number: 5382000015)
  7. NanoDrop OneC (Thermo Fisher Scientific, catalog number: ND-ONEC-W)
  8. Odyssey CLX Imaging System (Li-Cor)
  9. PowerPac HV High-Voltage power supply (Bio-Rad, catalog number: 1645056)
  10. PowerPac HC High-Current power supply (Bio-Rad, catalog number: 1645052)
  11. Owl HEP Series semidry electroblotting system (Thermo Fisher Scientific, catalog number: HEP-1)
  12. ImageQuant 300 imager (GE Healthcare, catalog number: 63-0056-52)
  13. UV Transilluminator (Fotodyne, catalog number: 3-3000)
  14. UV Crosslinker, 254 nm (Spectroline, catalog number: XL1000)
  15. Innova 2000 platform shaker (New Brunswick, catalog number: M1190-0000)
  16. Hybridization oven (VWR, model number: 2720)
  17. Mini-PROTEAN Tetra vertical electrophoresis system (Bio-Rad, catalog number: 1658005)
    Note: Although this system is designed for SDS-polyacrylamide gel electrophoresis (PAGE), we use it routinely to prepare and run Urea-PAGE for RNA separation.
  18. Tabletop centrifuge with refrigeration (Eppendorf, catalog number: 5404000138)
  19. -20 °C freezer (GE, model number: FUM21DHRWW)
  20. -80 °C freezer (New Brunswick, model number: C760)


  1. Image Studio (LiCor; free version available upon request at


  1. Preparation of IR-labeled probe
    1. Dissolve azide-containing DNA oligonucleotide with double distilled H2O (ddH2O). Final DNA concentration is 100 µM.
    2. Combine 2.5 nmole oligos with 50 nmole IRDye 680RD or 800CW DBCO in 1x phosphate buffered saline (PBS) in total volume of 50 µl.
      Note: From this point on, the probe should be kept away from light.
    3. Incubate reaction at room temperature for 6 h.
      Note: Incubation was performed without agitation/shaking. We have also incubated overnight with no loss of yield. However, we have not determined the minimal incubation time for sufficient labeling.
    4. Add 2 volumes of AMPure XP beads and 5.4 volumes of isopropanol; mix well by pipetting up and down 10 times.
    5. Incubate at room temperature for 7½ min. Remix, incubate for another 7½ min.
    6. Place beads on magnet stand. After beads settle, remove the supernatant. 
    7. Wash twice with freshly prepared 85% ethanol.
    8. Elute in 100 µl of ddH2O; move the eluate to a new tube.
    9. Use NanoDrop to measure 1 µl and determine DNA concentration. Typical recovery efficiency is 70-90%.

  2. Alternative probe preparation I–prepare DNA probe using terminal transferase
    1. Prepare 30 µl terminal transferase (TdT) reaction in a 1.5 ml centrifuge tube with 1x Terminal Transferase Reaction Buffer, 0.25 mM CoCl2, 100 pmoles of DNA oligo (no internal azide), 300 pmoles of 5-Azidomethyl-dUTP, 20 U of TdT, and ddH2O.
      Note: Reaction can be scaled up for preparing a large batch of probes. However, we recommend small scale TdT reaction with different probe:5-Azidomethyl-dUTP ratios to estimate labeling efficiency first.
    2. Incubate reaction at 37 °C for 1 h in ThermoMixer C.
      Note: Incubation was performed without agitation/shaking.
    3. Follow Steps A4-A7 to purify the DNA using AMPure XP beads.
    4. Elute in 20 µl of ddH2O; move the eluate to a new tube.
    5. Measure DNA concentration by loading 1 µl on a NanoDrop.
    6. Label 19 µl of probe with 10 nmole IRDyes 680RD or 800CW DBCO in 1x PBS diluted in ddH2O.
      Note: Make sure the molar ratio between IR dye and probe is greater than 10:1.
    7. Incubate at room temperature for 6 h.
    8. Follow Steps A4-A7 to purify the probe using AMPure XP beads.
    9. Elute in 15 µl of ddH2O. Run entire IR-labeled oligo eluate on an 8 M urea 15% polyacrylamide gel and run 10 pmoles of unlabeled oligo.
      Note: It is important to run unlabeled probe to estimate how many nucleotides have been added by TdT reaction.
    10. Detect IR signal on Li-Cor Odyssey CLX Scanner (Figure 1A).
    11. Stain the gel with 1x SYBR Green I and visualize the signal in ImageQuant 300 Imager (Figure 1A).
    12. Match IR image and SYBR Green I staining image to determine the most prominently-labeled probes. 
    13. Excise the gel slice containing the most prominently-labeled probes.
    14. Place gel pieces in a 1.5 ml centrifuge tube and elute overnight at room temperature in 600 µl 1:1 RNA Elution Buffer:Phenol/Chloroform/Isoamyl Alcohol with gentle rotation on a rotating mixer.
    15. Transfer the liquid to a new 1.5 ml tube.
    16. Vortex for 30 s and spin at 21,000 x g at 4 °C for 5 min.
    17. Transfer the aqueous phase to a new 1.5 ml centrifuge tube.
    18. Add 300 µl of chloroform, vortex for 30 s, and spin at 21,000 x g at 4 °C for 5 min.
    19. Transfer the aqueous phase to a new 1.5 ml centrifuge tube.
      Note: We recommend adding 15 µg of glycogen to co-precipitate with the DNA and help visualize the pellet.
    20. Add 1 ml cold 100% ethanol and incubate at -80 °C for 15 min.
    21. Spin at 21,000 x g for 30 min at 4 °C.
    22. Remove all the solution and air-dry the pellet for ~3 min or until dry.
    23. Resuspend RNA in 22 µl of ddH2O.
    24. Quantify 1 µl of RNA using NanoDrop.

  3. Alternative Probe Preparation II–generate RNA probes using T7 run-off Transcription
    1. Design and prepare PCR templates containing a T7 promoter to be used in a T7 run-off transcription reaction.
    2. Incubate overnight at 37 °C a 10 µl reaction containing 40 mM Tris-HCl pH 8.0, 25 mM NaCl, 2 mM Spermidine (HCl)3, 8 mM MgCl2, 1 mM ATP, 1 mM total UTP (3:1 ratio of UTP:5-azido-C3-UTP), 1 mM GTP, 1 mM CTP, 20 U Murine RNase Inhibitor, 10 mM DTT, and 4 U T7 RNA polymerase.
      Note: Reaction can be scaled up for preparing a large batch of probes. However, we recommend several small scale T7 in vitro transcriptions with different UTP:5-azido-C3-UTP ratios to estimate transcription and subsequent IR dye labeling efficiency first.
    3. Separate the in vitro transcribed RNAs using an 8 M urea 6% polyacrylamide gel and detect the RNA by UV shadowing, followed by excision of the gel slice containing the RNA.
    4. Place the gel slice in a 1.5 ml centrifuge tube and elute overnight at room temperature in 600 µl 1:1 RNA Elution Buffer:Phenol/Chloroform/Isoamyl Alcohol with gentle rotation on a rotating mixer.
    5. Follow Steps B15-B24 to extract and ethanol precipitate the RNA.
    6. Label RNA probe with IRDye 680RD or 800CW DBCO as described in Steps B6-B7.
    7. Purify by AMPure XP beads as described in Steps A4-A7.
    8. Elute in 15 µl of ddH2O. Run entire IR-labeled RNA eluate on an 8 M urea 6% polyacrylamide gel.
    9. Detect IR signal on Li-Cor Odyssey CLX Scanner (Figure 1B).
    10. Stain the gel with 1x SYBR Green I and visualize the signal with ImageQuant 300 Imager (Figure 1B).
    11. Excise the labeled probes.
    12. Place gel pieces in a 1.5 ml centrifuge tube and elute overnight at room temperature in 600 µl 1:1 RNA Elution Buffer:Phenol/Chloroform/Isoamyl Alcohol with gentle rotation on rotating mixer.
    13. Follow Steps B15-B24 to extract and ethanol precipitate the RNA probe.

  4. TRIzol extraction of RNA
    1. Culture HCT116 and HCT116 Drosha knockout cells in a 6-well plate in McCoy’s 5A Media with 10% fetal bovine serum and 1% penicillin and streptomycin.
    2. When confluent, remove media and rinse cells with 1x PBS. Collect cells in a 1.5 ml microcentrifuge tubes and pellet at 1,000 x g for 3 min at 4 °C.
    3. Remove bulk of the supernatant, briefly resuspend cells in the residual (~20-30 µl) 1x PBS.
      Note: If TRIzol is added to a dry cell pellet, the cells will not lyse completely.
    4. Add 1 ml of TRIzol to each cell pellet collected from one well and vortex for 30 s.
    5. Add 200 µl of chloroform, vortex for 30 s and spin at 12,000 x g for 15 min at 4 °C.
    6. Transfer the aqueous phase (about 550 µl) to a new 1.5 ml microcentrifuge tube.
    7. Precipitate the RNA by adding equal volume (550 µl) of isopropanol and incubate at room temperature for 10 min.
    8. Spin at 21,000 x g for 20 min at 4 °C, a white pellet should form at the bottom of the tube.
    9. Remove the supernatant and wash the pellet with 1 ml of cold 75% ethanol.
    10. Spin at 21,000 x g for 5 min at 4 °C.
    11. Remove supernatant, air-dry pellet for ~3 min, and resuspend RNA in 20 µl of ddH2O.
    12. Mix 1 µl of RQ1 DNase with 5 µl 10x DNase RQ1 buffer and 24 µl ddH2O and add mix to RNA.
    13. Incubate reaction at 37 °C for 30 min.
    14. Bring volume to 300 µl with ddH2O and add 300 µl of Phenol/Chloroform/Isoamyl Alcohol.
    15. Follow Steps B16-B24 to extract and ethanol precipitate the total RNA. Glycogen could be omitted.

  5. Urea-PAGE, transfer, and Northern blot
    1. Mix 15-20 µg of total RNA sample with equal volume of 2x Formamide Loading Dye, heat samples at 95 °C for 3 min, immediately place on ice, and separate RNA on an 8 M urea 15% polyacrylamide gel.
    2. Transfer the gel at 300 mAmps for 1 h to Hybond N+ membrane using Owl HEP Series semidry electroblotting system.
      Note: Alternatively, RNAs separated by Urea-PAGE could be transferred using wet-transfer system (RNA: a laboratory manual, Chapter 3, Protocol 11). If RNAs were separated on an agarose gel, traditional capillary transfer method should be used (RNA: a laboratory manual, Chapter 3, Protocol 9).
    3. Briefly dry membrane on a paper towel and crosslink membrane twice using 254 nm UV crosslinker at 120 mJ/cm2.
    4. In the hybridization oven, pre-hybridize the membrane in a hybridization tube with 10 ml ExpressHyb hybridization solution for 30 min at 30 °C.
    5. Remove the pre-hybridization solution and hybridize overnight at 30 °C with 10 pmoles IR probe diluted in 10 ml ExpressHyb hybridization solution.
      1. If different probes have been labeled with different IRDyes, they can be combined together to detect different RNAs simultaneously.
      2. Hybridization temperature should be adjusted for different probes, ~20 °C below calculated Tm. We recommend a hybridization temperature of 30 °C for miRNAs. When using RNA probes, we typically perform hybridization at 65 °C overnight, with no loss of signal due to temperature.
    6. Collect diluted probe in a 15 ml centrifuge tube and store at -20 °C.
      Note: Probes can be reused several times without significant loss of signal.
    7. Place membrane in a clean glass tray and add 2x SSC (Saline-sodium citrate)-0.1% SDS Wash Buffer to wash the membrane.
      Note: Submerge the membrane under ~1 cm solution.
    8. Shake at 110 rpm for 10 min at room temperature.
      Note: Washing temperature can be adjusted for different probes, depending on the Tm between the probe and the target.
    9. Perform second wash with 1x SSC-0.1% SDS Wash Buffer for 10 min at room temperature with shaking at 110 rpm.
      Note: If higher washing stringency is desired, continue with washing buffer containing lower SSC concentration and higher SDS concentration.
    10. Briefly tap dry the membrane with paper towels.
    11. Image on Li-Cor Odyssey CLX Scanner to detect emission at 700 nm and/or 800 nm (Figure 1C).
    12. To strip the probes off the membrane; place membrane in a glass tray, pour microwave boiled 0.1x SSC-1% SDS-40 nM Tris-HCl pH 8.0, shake at 110 rpm for 10 min at room temperature.
    13. Repeat strip procedure (Step E12), dry membrane with paper towels, and store in sheet protector.
      Note: Membrane can be stored for years and re-probed later.

Data analysis

Data is analyzed using the Image Studio software. Users can adjust brightness and contrast without changing the original image. The software also allows for the quantitation of signal, although we did not do it in this protocol. For users who want to multiplex, the software simultaneously scans at two separate wavelengths and overlays the images, generating two independent but perfectly superimposed images.

Figure 1. Alternative generation of IR probes and detection of let-7a and U6 using IR probes. A. DNA IR-probes generated from terminal transferase reactions and separated on an 8 M urea 15% polyacrylamide gel. The gel was scanned on a Li-Cor Odyssey CLX Scanner and then stained with SYBR Green I before being visualized using a UV transilluminator. The bands containing the desired IR-labeled probe are indicated by brackets. B. RNA IR-probe generated by T7 in vitro transcription and separated on an 8 M urea 6% polyacrylamide gel. The gel was scanned on the Li-Cor Odyssey CLX Scanner, stained with SYBR Green I, and then visualized on a UV transilluminator. The IR-labeled RNA was excised. C. irNorthern simultaneously detects miRNA let-7a and small nuclear RNA U6. Total RNA from HCT116 wild type and Drosha Knockout (KO) cells were extracted using TRIzol, separated on an 8 M urea 15% polyacrylamide gel, and transferred to a membrane. irNorthern probes for let-7a and U6 were hybridized overnight and the membrane was visualized using the Li-Cor Odyssey CLX Scanner. Canonical miRNAs (i.e., let-7a) require Drosha for processing and are not expressed in Drosha KO cells (Kim et al., 2016). U6 is utilized as a loading control. Images in Figures 1A and 1B were originally published in RNA under Miller et al., 2018 and can be found at


  1. When working with IR dyes, it is important to keep stocks away from light using aluminum foil wrapped tubes or amber tubes. Additionally, we have found that IRDye 800CW-labeled probes are more sensitive than 680RD-labeled probes. Therefore, we suggest for multiplexing, using the 800CW-labeled probes for less abundant targets.
  2. RNA probes generated in Procedure C cannot be completely stripped from the membrane after hybridization to the target RNAs.


  1. 1x Phosphate Buffered Saline (PBS) (pH 7.4)
    137 mM NaCl
    10 mM Na2HPO4
    2.7 mM KCl 
  2. RNA Elution Buffer
    300 mM NaOAc
    25 mM Tris-HCl, pH 8.0
  3. 8 M urea 15% polyacrylamide gel
    5 g Urea
    2 ml 5x TBE (Tris-Borate-EDTA)
    625 µl ddH2O
    *Microwave until the solution is warm (~5-8 s)
    3.75 ml 40% Acrylamide
    *Rotate until urea completely dissolved
    52 µl 10% APS
    10 µl TEMED
  4. 8 M urea 6% polyacrylamide gel
    5 g Urea
    2 ml 5x TBE
    2.875 ml ddH2O
    *Microwave until the solution is warm (~5-8 s)
    1.5 ml 40% Acrylamide
    *Rotate until urea completely dissolved
    52 µl 10% APS
    10 µl TEMED
  5. 2x Formamide loading Dye
    80% Formamide
    0.1% Bromophenol Blue
    0.1% Xylene Cyanol
    20 mM EDTA
    20 mM Tris-HCl, pH 8.0


The Xie Lab is supported by the National Institute of Health (R00-CA190886 and R35-GM128753 to M.X.), University of Florida Research Opportunity Seed Fund (to M.X.), and Thomas H. Maren Foundation (Junior Investigator Fund F013372 to M.X.).

Competing interests

The authors declare no conflict of interest or competing interest.


  1. Alwine, J. C., Kemp, D. J. and Stark, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 74(12): 5350-5354.
  2. Jones, C. G. (2005). A review of the history of U.S. radiation protection regulations, recommendations, and standards. Health Phys 88(2): 105-124.
  3. Kim, Y. K., Kim, B. and Kim, V. N. (2016). Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis. Proc Natl Acad Sci U S A 113(13): E1881-1889.
  4. Miller, B. R., Wei, T., Fields, C. J., Sheng, P. and Xie, M. (2018). Near-infrared fluorescent Northern blot. RNA 24(12): 1871-1877.
  5. Rio, D.C., Hannon, G.J., Ares Jr., M., and Nilsen, T.W. (2011). RNA: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press. ISBN: 978-0-879698-91-1.
  6. Seibl, R., Holtke, H. J., Ruger, R., Meindl, A., Zachau, H. G., Rasshofer, R., Roggendorf, M., Wolf, H., Arnold, N., Wienberg, J. and et al. (1990). Non-radioactive labeling and detection of nucleic acids. III. Applications of the digoxigenin system. Biol Chem Hoppe Seyler 371(10): 939-951.
  7. Zarnegar, B. J., Flynn, R. A., Shen, Y., Do, B. T., Chang, H. Y. and Khavari, P. A. (2016). irCLIP platform for efficient characterization of protein-RNA interactions. Nat Methods 13(6): 489-492.


Northern blot是一种分子生物学技术,可以检测,定量和确定RNA的分子量。 最近,我们在Northern blot(irNorthern)中发表了一种利用近红外(IR)荧光探针的方案。 我们的方法与其他非放射性方法一样敏感,但更直接,更通用。 此外,我们发现IR标记的探针可以同时用于多重或检测不同种类的RNA。 在这里,我们描述了三种生成IR标记探针的方法以及如何进行irNorthern印迹。 总之,我们的irNorthern协议提供了一种方便的RNA检测方法。
【背景】Northern印迹分析是生物化学家和分子生物学家可用的基础技术之一。虽然采用了更新和更高通量的技术,但Northern印迹仍然是同时分析RNA大小和数量的强大且适应性强的方法。简而言之,用放射性磷-32( 32 P)标记的DNA或RNA寡核苷酸与用RNA交联的膜杂交,随后使用放射自显影检测(Alwine et al。, 1977年)。尽管如此, 32 P的使用受制度监管,在使用过程中必须遵守安全预防措施(Jones,2005)。此外, 32 P探针寿命短,增加了使用成本。已经开发了使用与dNTP或NTP缀合的洋地黄毒苷(DIG)的替代方案来代替 32 P(Seibl 等人,,1990)。虽然成功,但DIG探针需要通过抗体进行二次检测,更多的手工操作时间和昂贵的试剂,阻碍了其更广泛的应用。我们最近发布的方法irNorthern很简单,适应性强。它利用IR染料降低自发荧光背景(Zarnegar et al。,2016; Miller et al。,2018)。这些IR染料与二苯并环辛炔基团(DBCO)共轭,其通过无铜点击化学与探针上的叠氮化物形成共价键。为了证明其应用,我们进行了irNorthern检测从HCT116野生型和Drosha敲除细胞中提取的总RNA中的microRNA let-7a和小核RNA U6(Kim et al。,2016)。此外,我们提供了有关如何生成三种不同的含叠氮基的DNA或RNA探针用于IR染料标记的详细信息:化学合成的DNA探针,在dT上具有内部叠氮化物修饰,DNA探针通过末端转移酶用叠氮化物-dU标记( TdT)和用T7 RNA聚合酶用叠氮化物-U标记的RNA探针。

关键字:Northern杂交, 近红外, irNorthern, RNA检测, 非放射性的


  1. Seal-Rite 1.5 ml微量离心管(USA Scientific,目录号:1615-5599)
  2. 杂交管(Fisher Scientific,目录号:13-247-300)
  3. Hybond N + 30 cm x 3 M Roll(GE,目录号:RPN303B)
  4. 50毫升离心管(Genesee,目录号:21-106)
  5. 15毫升离心管(Genesee,目录号:21-101)
  6. 1,250μl移液器吸头(USA Scientific,目录号:1112-1720)
  7. 0.1-10 /20μl移液器吸头(USA Scientific,目录号:1110-3700)
  8. 200μl移液器吸头(USA Scientific,目录号:1110-1700)
  9. Whatman Grade 3 MM Chr吸墨纸,46 x 57 cm(GE,目录号:3030-917)
  10. 6孔细胞培养板(Genesee Scientific,目录号:25-105)
  11. 纸巾(Highmark,目录号4497A1)
  12. 玻璃托盘(耐热玻璃,目录号:1124847)
  13. 铝箔卷(Fisher,目录号:01-213-102)
  14. 板材保护装置(Office Depot,目录号:491694)
  15. HCT116亲本细胞系(韩国典型培养物保藏中心,目录号:BP1230983)
  16. HCT116 Drosha KO#40细胞系(韩国典型培养物保藏中心,目录号:BP1230984)
  17. let-7a irNorthern探针(5'-AACTATACAACCTACTACCTCA / iAzideN / A-3')
    1. 探针以100 nmole规模从Integrated DNA Technology订购,含有一个内部叠氮化物,并通过HPLC纯化。
    2. DNA探针设计为与靶RNA互补。对于miRNA靶标,探针是成熟miRNA的全长(~22nt)反向互补序列。根据需要,在3'末端附近添加内部叠氮化物和末端核苷酸用于合成。
  18. U6 irNorthern探测器(5'-GCAGGGGCCATGCTAATCTTCTCTGTATCG / iAzideN / T-3')
    注意:常用靶RNA的探针序列可在文献中找到。在设计新的DNA探针时,我们建议使用~30 nt序列靶向RNA的单链区域。
  19. 末端转移酶(NEB,目录号:M0315)
  20. 5-叠氮甲基-dUTP(Jena Biosciences,目录号:CLK-084S)
  21. 5-Azido-C 3 -UTP(Jena Biosciences,目录号:NU-157S)
  22. Agencourt AMPure XP珠子(Beckman Coulter,目录号:A63881)
  23. 异丙醇(J.T.Baker,目录号:908401)
  24. 乙醇(Decon Laboratories,目录号:2716)
  25. IRDye 680RD DBCO红外染料,0.5 mg(Li-Cor,目录号:929-50005) - 光敏感
  26. IRDye 800CW DBCO红外染料,5毫克(Li-Cor,目录号:929-55000) - 光敏感
  27. 尿素(西格玛,目录号:U5378)
  28.   Acryl / Bis 19:1(VWR,目录号:97064-990)
  29. 过硫酸铵(APS)(AmericanBio,目录号:AB00112)
  30. TEMED(N,N,N',N'-四甲基乙二胺)(AmericanBio,目录号:AB02020)
  31. 氯化钠(AmericanBio,目录号:AB01915)
  32. 无水磷酸氢二钠(Fisher Scientific,目录号:BP332-500)
  33. 氯化钾(Sigma,目录号:P9541)
  34. 醋酸钠(Sigma,目录号:S7545)
  35. Trizma base(Sigma,目录号:T1503)
  36. HEPES(Sigma,目录号:H4034)
  37. 乙二胺四乙酸(EDTA)(Sigma,目录号:EDS)
  38. 十二烷基硫酸钠(SDS)(AmericanBio,目录号:AB01920)
  39. Formamide DI(AmericanBio,目录号:AB00600-00500)
  40. 溴酚蓝(Sigma,目录号:114391)
  41. 二甲苯Cyanol FF(Sigma,目录号:X4126)
  42. 柠檬酸钠二水合物(Sigma,目录号:W302600)
  43. 苯酚/氯仿/异戊醇(Fisher Scientific,目录号:BP1752I-400)
  44. 氯仿(AmericanBio,目录号:AB00350-01000)
  45. ExpressHyb杂交解决方案(Clontech,目录号:636832)
  46. TRIzol(Ambion,目录号:15596018)
  47. SYBR Green I Nucleic Acid Gel Stain(Invitrogen,目录号:S7563)
  48. A,C,U,G核糖核苷酸(Thermo Fisher Scientific,目录号:R0481)
  49. 亚精胺三盐酸盐(Sigma,目录号:85578)
  50. RNase Inhibitor,Murine(NEB,目录号:M0314L)
  51. 二硫苏糖醇(DTT)(AmericanBio,目录号:AB00490-00025)
  52. 氯化镁六水合物(Sigma,目录号:M2670)
  53. HYCLN McCoys 5A Media(Fisher Scientific,目录号:SH30200)
  54. 胎牛血清(PAA Laboratories,目录号:A15-201)
  55. 青霉素 - 链霉素(Thermo Fisher Scientific,目录号:15070063)
  56. RQ1 RNase-Free DNase(Promega,目录号:M6101)
  57. 1x磷酸盐缓冲盐水(PBS)(见食谱)
  58. RNA洗脱缓冲液(见食谱)
  59. 8 M尿素15%聚丙烯酰胺凝胶(见食谱)
  60. 8 M尿素6%聚丙烯酰胺凝胶(见食谱)
  61. 2x甲酰胺装载染料(见食谱)


  1. 16管SureBeads磁力架(Bio-Rad,目录号:1614916)
  2. Vortex Genie 2(Fisher Scientific,目录号:12-812)
  3. 微量移液器(Eppendorf,Research Plus Models)
  4. Roto-Bot旋转混合器(Benchmark Scientific,目录号:R4045)
  5. Water-Jacketed CO 2 培养箱(Forma Scientific,目录号:3110)
  6. ThermoMixer C (Eppendorf,目录号:5382000015)
  7. NanoDrop OneC(赛默飞世尔科技,目录号:ND-ONEC-W)
  8. Odyssey CLX成像系统(Li-Cor)
  9. PowerPac HV高压电源(Bio-Rad,目录号:1645056)
  10. PowerPac HC大电流电源(Bio-Rad,目录号:1645052)
  11. 猫头鹰HEP系列半干式电印迹系统(赛默飞世尔科技,目录号:HEP-1)
  12. ImageQuant 300成像仪(GE Healthcare,目录号:63-0056-52)
  13. UV Transilluminator(Fotodyne,目录号:3-3000)
  14. UV交联剂,254 nm(Spectroline,目录号:XL1000)
  15. Innova 2000平台振动筛(New Brunswick,目录号:M1190-0000)
  16. 杂交烤箱(VWR,型号:2720)
  17. Mini-PROTEAN Tetra立式电泳系统(Bio-Rad,目录号:1658005)
  18. 带冷藏的台式离心机(Eppendorf,目录号:5404000138)
  19. -20°C冰箱(GE,型号:FUM21DHRWW)
  20. -80°C冰箱(新不伦瑞克省,型号:C760)


  1. Image Studio(LiCor;免费版本可根据licor.com的要求提供)


  1. 制备IR标记的探针
    1. 用双蒸馏的H 2 O(ddH 2 O)溶解含叠氮基的DNA寡核苷酸。最终DNA浓度为100μM。
    2. 将2.5 nmole寡核苷酸与50 nmole IRDye 680RD或800CW DBCO在1x磷酸盐缓冲盐水(PBS)中混合,总体积为50μl。
    3. 在室温下孵育反应6小时。
    4. 加入2倍体积的AMPure XP珠粒和5.4倍体积的异丙醇;通过上下移液10次混合均匀。
    5. 在室温下孵育7½分钟。混合,再孵育7½分钟。
    6. 将珠子放在磁铁架上。珠子沉淀后,去除上清液。 
    7. 用新鲜配制的85%乙醇洗两次。
    8. 在100μlddH 2 O中洗脱;将洗脱液移至新管中。
    9. 使用NanoDrop测量1μl并确定DNA浓度。典型的回收效率为70-90%。

  2. 替代探针制备I-使用末端转移酶制备DNA探针
    1. 在1.5 ml离心管中准备30μl末端转移酶(TdT)反应,其中含有1x末端转移酶反应缓冲液,0.25 mM CoCl 2 ,100 pmoles DNA oligo(无内部叠氮化物),300 pmoles of 5- Azidomethyl-dUTP,20 U TdT和ddH 2 。
    2. 在ThermoMixer C中37℃孵育反应1小时。
    3. 按照步骤A4-A7使用AMPure XP珠子纯化DNA。
    4. 在20μlddH 2 O中洗脱;将洗脱液移至新管中。
    5. 通过在NanoDrop上加载1μl来测量DNA浓度。
    6. 在稀释于ddH 2 O的1x PBS中标记19μl具有10nmole IRDyes 680RD或800CW DBCO的探针。
    7. 在室温下孵育6小时。
    8. 按照步骤A4-A7使用AMPure XP珠子纯化探针。
    9. 在15μlddH 2 O中洗脱。在8 M尿素15%聚丙烯酰胺凝胶上运行完整的IR标记的寡聚洗脱液,并运行10 pmoles未标记的寡核苷酸。
    10. 检测Li-Cor Odyssey CLX扫描仪上的红外信号(图1A)。
    11. 用1x SYBR Green I染色凝胶,并在ImageQuant 300 Imager中观察信号(图1A)。
    12. 匹配红外图像和SYBR Green I染色图像,以确定最突出标记的探针。 
    13. 切除含有最突出标记探针的凝胶切片。
    14. 将凝胶块置于1.5ml离心管中,在室温下在600μl1:1 RNA洗脱缓冲液:苯酚/氯仿/异戊醇中洗脱过夜,在旋转混合器上轻轻旋转。
    15. 将液体转移到新的1.5毫升管中。
    16. 涡旋30秒并在4℃下以21,000 x g 旋转5分钟。
    17. 将水相转移到新的1.5ml离心管中。
    18. 加入300μl氯仿,涡旋30秒,并在4℃下以21,000 x g 旋转5分钟。
    19. 将水相转移到新的1.5 ml离心管中。
    20. 加入1ml冷的100%乙醇并在-80℃下孵育15分钟。
    21. 在4℃下以21,000 x g 旋转30分钟。
    22. 取出所有溶液,风干颗粒约3分钟或直至干燥。
    23. 在22μlddH 2 O中重悬RNA。
    24. 使用NanoDrop定量1μlRNA。

  3. 替代探针制备II-使用T7径流转录产生RNA探针
    1. 设计并制备含有T7启动子的PCR模板,用于T7径流转录反应。
    2. 在37℃下孵育过夜,含有40mM Tris-HCl pH 8.0,25mM NaCl,2mM亚精胺(HCl) 3 ,8mM MgCl 2 的10μl反应, 1 mM ATP,1 mM总UTP(UTP比例为3:1:5-azido-C 3 -UTP),1 mM GTP,1 mM CTP,20 U鼠RNase抑制剂,10 mM DTT和4 U T7 RNA聚合酶。
      注意:可以按比例放大反应以准备大批量的探针。然而,我们建议使用不同的UTP:5-azido-C 3 -UTP比率的几种小规模T7体外转录来估计转录和随后的IR染料标记效率优先。
    3. 使用8M尿素6%聚丙烯酰胺凝胶分离体外转录的RNA,并通过UV遮蔽检测RNA,然后切除含有RNA的凝胶切片。
    4. 将凝胶切片置于1.5ml离心管中,在室温下在600μl1:1 RNA洗脱缓冲液:苯酚/氯仿/异戊醇中洗脱过夜,在旋转混合器上轻轻旋转。
    5. 按照步骤B15-B24提取并乙醇沉淀RNA。
    6. 如步骤B6-B7所述,用IRDye 680RD或800CW DBCO标记RNA探针。
    7. 如步骤A4-A7中所述,通过AMPure XP珠粒纯化。
    8. 在15μlddH 2 O中洗脱。在8M尿素6%聚丙烯酰胺凝胶上运行完整的IR标记的RNA洗脱液。
    9. 检测Li-Cor Odyssey CLX扫描仪上的红外信号(图1B)。
    10. 用1x SYBR Green I染色凝胶,用ImageQuant 300 Imager观察信号(图1B)。
    11. 切除标记的探针。
    12. 将凝胶块置于1.5ml离心管中,在室温下在600μl1:1 RNA洗脱缓冲液:苯酚/氯仿/异戊醇中洗脱过夜,在旋转混合器上轻轻旋转。
    13. 按照步骤B15-B24提取并用乙醇沉淀RNA探针。

  4. TRIzol提取RNA
    1. 在具有10%胎牛血清和1%青霉素和链霉素的McCoy 5A培养基中的6孔板中培养HCT116和HCT116 Drosha敲除细胞。
    2. 汇合时,取出培养基并用1x PBS冲洗细胞。将细胞收集在1.5ml微量离心管中,并在4℃下以1,000 xg /小时沉淀3分钟。
    3. 去除大部分上清液,在残留的(~20-30μl)1x PBS中短暂重悬细胞。
    4. 向从一个孔收集的每个细胞沉淀中加入1ml TRIzol并涡旋30秒。
    5. 加入200μl氯仿,涡旋30秒,并在4℃下以12,000 x g 旋转15分钟。
    6. 将水相(约550μl)转移到新的1.5ml微量离心管中。
    7. 通过加入等体积(550μl)异丙醇沉淀RNA,并在室温下孵育10分钟。
    8. 在4℃下以21,000 x g 旋转20分钟,应在管底部形成白色颗粒。
    9. 除去上清液,用1ml冷的75%乙醇洗涤沉淀。
    10. 在4℃下以21,000 x g 旋转5分钟。
    11. 除去上清液,风干沉淀约3分钟,并将RNA重悬于20μlddH 2 O中。
    12. 将1μlRQ1DNA酶与5μl10xDNase RQ1缓冲液和24μlddH 2 O混合并向RNA中加入混合物。
    13. 在37°C孵育反应30分钟。
    14. 用ddH 2 O使体积达到300μl,并加入300μl苯酚/氯仿/异戊醇。
    15. 按照步骤B16-B24提取并用乙醇沉淀总RNA。糖原可以省略。

  5. 尿素-PAGE,转移和Northern印迹
    1. 将15-20μg总RNA样品与等体积的2x甲酰胺加载染料混合,在95℃加热样品3分钟,立即置于冰上,并在8M尿素15%聚丙烯酰胺凝胶上分离RNA。
    2. 使用Owl HEP系列半干电印迹系统将凝胶以300 mAmps转移至Hybond N +膜1小时。
    3. 在纸巾上短暂干燥膜并使用254nm UV交联剂在120mJ / cm 2下交联膜两次。
    4. 在杂交炉中,将杂交管中的膜与10ml ExpressHyb杂交溶液在30℃下预杂交30分钟。
    5. 去除预杂交溶液,并在30°C下与10 pmole杂交溶液稀释的10 pmoles IR探针杂交过夜。
      1. 如果不同的探针用不同的IRDye标记,它们可以组合在一起同时检测不同的RNA。
      2. 应针对不同的探针调整杂交温度,比计算的T m 低约20°C。我们建议miRNA的杂交温度为30°C。当使用RNA探针时,我们通常在65°C下进行杂交过夜,不会因温度而失去信号。
    6. 将稀释的探针收集在15 ml离心管中,并储存在-20°C。
    7. 将膜置于干净的玻璃盘中,加入2x SSC(盐水 - 柠檬酸钠)-0.1%SDS洗涤缓冲液洗涤膜。
      注意:将膜浸入~1 cm溶液中。
    8. 在室温下以110rpm摇动10分钟。
      注意:根据探头和目标之间的T m ,可以根据不同探头调整洗涤温度。
    9. 用1x SSC-0.1%SDS洗涤缓冲液在室温下以110rpm振荡进行第二次洗涤10分钟。
    10. 用纸巾轻轻擦干膜。
    11. Li-Cor Odyssey CLX扫描仪上的图像,用于检测700 nm和/或800 nm的发射(图1C)。
    12. 从膜上剥离探针;将膜置于玻璃盘中,倒入微波煮沸的0.1x SSC-1%SDS-40nM Tris-HCl pH 8.0,在室温下以110rpm摇动10分钟。
    13. 重复剥离程序(步骤E12),用纸巾擦干膜,并存放在保护膜中。


使用Image Studio软件分析数据。用户无需更改原始图像即可调整亮度和对比度。该软件还允许定量信号,尽管我们没有在该协议中进行。对于想要多路复用的用户,软件同时扫描两个不同的波长并覆盖图像,生成两个独立但完美叠加的图像。

图1.使用IR探针替代生成IR探针和检测let-7a和U6。 A.从末端转移酶反应产生的DNA IR探针,并在8 M尿素15%聚丙烯酰胺凝胶上分离。在Li-Cor Odyssey CLX扫描仪上扫描凝胶,然后用SYBR Green I染色,然后使用UV透射仪观察。含有所需IR标记探针的条带用括号表示。 B.通过T7 体外转录产生的RNA IR-探针,并在8M尿素6%聚丙烯酰胺凝胶上分离。在Li-Cor Odyssey CLX扫描仪上扫描凝胶,用SYBR Green I染色,然后在UV透射仪上显现。切下IR标记的RNA。 C. irNorthern同时检测miRNA let-7a和小核RNA U6。使用TRIzol提取来自HCT116野生型和Drosha敲除(KO)细胞的总RNA,在8M尿素15%聚丙烯酰胺凝胶上分离,并转移至膜。将let-7a和U6的irNorthern探针杂交过夜,并使用Li-Cor Odyssey CLX扫描仪显现膜。 Canonical miRNAs( ie。,let-7a)需要Drosha进行处理,并且不在Drosha KO细胞中表达(Kim et al。,2016)。 U6用作加载控制。图1A和1B中的图像最初是在Miller et al。,2018年的RNA中发表的,可以在


  1. 使用红外染料时,使用铝箔包裹的管或琥珀色管来保持库存远离光线非常重要。此外,我们发现IRDye 800CW标记的探针比680RD标记的探针更敏感。因此,我们建议使用800CW标记的探针进行多重化,以获得不太丰富的靶标。
  2. 在与靶RNA杂交后,在方法C中产生的RNA探针不能从膜上完全剥离。


  1. 1x磷酸盐缓冲盐水(PBS)(pH 7.4)
    137 mM NaCl
    10mM Na 2 HPO 4
    2.7 mM KCl 
  2. RNA洗脱缓冲液
    300 mM NaOAc
    25mM Tris-HCl,pH 8.0
  3. 8 M尿素15%聚丙烯酰胺凝胶
    625μlddH 2 O
  4. 8 M尿素6%聚丙烯酰胺凝胶
    2.875 ml ddH 2 O
  5. 2x Formamide装载染料
    20 mM EDTA
    20mM Tris-HCl,pH 8.0


谢实验室由国立卫生研究院(R00-CA190886和R35-GM128753至M.X.),佛罗里达大学研究机会种子基金(至M.X.)和Thomas H. Maren基金会(初级研究基金F013372至M.X.)提供支持。




  1. Alwine,J.C.,Kemp,D.J。和Stark,G.R。(1977)。 通过转移至重氮苄氧基甲基纸并与DNA探针杂交检测琼脂糖凝胶中特定RNA的方法。 Proc Natl Acad Sci USA 74(12):5350-5354。
  2. Jones,C。G.(2005)。 审查美国辐射防护法规,建议和标准的历史。 Health Phys 88(2):105-124。
  3. Kim,Y.K.,Kim,B。和Kim,V。N.(2016)。 重新评估 DROSHA , Exportin 5的角色和 DICER 在microRNA biogenesis中。 Proc Natl Acad Sci USA 113(13):E1881-1889。
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引用:Fields, C., Sheng, P., Miller, B., Wei, T. and Xie, M. (2019). Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation. Bio-protocol 9(8): e3219. DOI: 10.21769/BioProtoc.3219.