Introduction

Increased awareness and interest in the regulatory roles of noncoding small RNA have led to the modification of several existing technologies, such as real-time PCR and nuclease protection assays, to detect low levels of small RNA in a better way^{1, 2}. In addition, high-throughput methods such as those employing small RNA cDNA cloning, deep sequencing and microarray expression analysis have greatly accelerated the discovery and characterization of novel small RNA³. In comparison with these, conventional northern blot (also known as RNA gel blot) analysis is less sensitive and lower throughput. However, it is unmatched in its ability to display the size of small RNA accurately and can simultaneously display the sizes and amounts of multiple small RNAs that share significant sequence identity. For example, both the mature (20 nt) and precursor (70 nt) forms of microRNA (miRNA) can be simultaneously detected and such a pattern provides strong support for the contention that one's sequence is indeed a miRNA⁴.

As size and size complexity are critical parameters in the validation and functional characterization of novel small RNA, northern blotting has remained a popular and valuable analytical method.

Northern blot analysis of small RNA involves the separation of RNA molecules according to size using denaturing PAGE (dPAGE) followed by transfer of the separated molecules onto, typically, a nylon membrane. The transferred molecules are usually cross-linked to the membrane using UV irradiation to reduce loss of the sample RNA during subsequent hybridization and wash steps, in which specific labeled nucleic acid probes complementary to the RNA sequence of interest are allowed to hybridize with the immobilized sample RNA. Other cross-linking methods such as baking at 80 °C or alkaline-assisted fixation are used for immobilizing RNA that are typically of mRNA size range but have not been widely used to cross-link small RNA.

Cross-linking with UV is rapid, inexpensive and works well for RNA >100 nt in length. It is thought that UV produces reactive-functional groups within the bases of RNA (principally Uridine), which then react with free amine groups on the nylon membrane surface to form covalent bonds^{5, 6, 7}. However, the evidence for this is indirect and the exact mechanism has not been demonstrated yet. If one presumes that the nucleotide bases are involved in forming the cross-link, then it is reasonable to think that this may reduce the subsequent availability of that base for hybridization with a complementary probe. Furthermore, as the number of bases involved in cross-linking cannot be controlled in the UV-triggered reaction, over-cross-linking might occur, hindering the accessibility of the probe for hybridization with the target sequence. We thought that such events may have an increasingly negative effect as the length of RNA to be detected decreased and so is particularly problematic for analyses of siRNA (short-interfering RNA) and miRNA. Our everyday laboratory experience of using northern blots to detect small RNA, such as siRNA and miRNA, with UV cross-linking had lead to numerous frustrations with lack of sensitivity and reproducibility. We could not improve the UV cross-linking procedure by varying UV dose and so we explored alternative methods for cross-linking. We adopted the premise that cross-linking would be optimal if all the nucleotides of a small RNA remained accessible for hybridization to a complementary probe. Therefore, we investigated cross-linking through the terminal ends of a small RNA. The RNase III-type nuclease activity involved in the production of small regulatory RNA, such as miRNA and siRNA, dictate a 5'-terminal monophosphate and a 2', 3' cis-diol at the 3'-terminus⁸. Different 3'-end modifications of small RNA have been described in mammals, insects and plants^{9, 10}, so we thought that cross-linking through the 5'-terminal monophosphate would provide the most flexible technique to immobilize many different small RNA.

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) has been used for cross-linking synthetic 5'-phosphorylated oligodeoxyribonucleotides to primary amines on different solid substrates and this resulted in enhanced hybridization compared with nitrocellulose-bound DNA^{11, 12, 13}. We tested whether EDC could be used for efficient cross-linking of small RNA from biological samples to commonly available nylon membrane (Hybond NX) following transfer from a dPAG and found that detection of small RNA isolated from plant or mammalian sources can be enhanced by up to 50-fold in comparison with UV cross-linking. Different regimes of hybridization conditions, including a range of different types of complementary probes, showed similar enhancement of detection after EDC cross-linking¹⁴. Our data was consistent with EDC facilitating the formation of a covalent phosphoramidate bond between 5'-terminal phosphates on small RNA and primary amine groups on the surface of the nylon membrane. Non-5'-phosphorylated RNA should also be detectable using this method if they are first phosphorylated (e.g., with T4 polynucleotide kinase). The enhanced detection we observed was significant for RNA molecules <40 nt in length. No great enhancement was detected for larger molecules, for example, miRNA precursors 70 nt (ref. 14). From this, we infer that the enhanced detection afforded by EDC cross-linking declines as the size of the RNA of interest increases. Thus, owing to its greater simplicity, UV cross-linking would remain the method of choice for RNA greater than 70 nt.

Here, we have described the small RNA northern blotting procedure as we use in it in our laboratory (see Fig. 1 for an overview of the protocol, or Supplementary Fig. 1 online, which provides more detail and can be used as a quick-reference protocol). We describe only the preparation of northern blots here and would suggest that subsequent hybridization of probes to these blots to detect specific RNAs be carried out using conventional probe preparation and hybridization conditions. There are many methods of both probe preparation and hybridization described in general molecular biology laboratory manuals, such as Sambrook and Russell¹⁵, or in information provided by commercial manufacturer(s) of the reagents used in northern blotting. In principle, any methods previously used for UV-cross-linked blots should be equally applicable for EDC-cross-linked blots.

Figure 1: Small RNA northern blotting protocol overview.

Chemical 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linking facilitates the production of a covalent bond between the terminal phosphate of the RNA to a free amino group on the nylon membrane resulting in the small RNA becoming tethered by one end. This allows the full sequence of the small RNA to be accessible to hybridizing probes in contrast to UV cross-linking which may lead to consumption of uridine residues during the cross-linking process and hence compromise the length of the sequence available for hybridization (see also Supplementary Fig. 1 for additional protocol details). dPAG, denaturing polyacrylamide gel. M, [³²P] ATP-labeled Decade marker.

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Experimental design

RNA sample material. A wide range of reagents and protocols to extract RNA, from plant or animal tissue, are available to isolate good-quality RNA. We typically use TRI-reagent (Sigma) but have successfully used the original Chomczynski and Sacchi protocol¹⁶, RNAbee (Biogenesis) and Trizol (Invitrogen) to isolate intact RNA, following manufacturer's recommended protocols. We have used total and low-molecular weight preparations of RNA (prepared as described by Hamilton and Baulcombe)¹⁷ and found that cross-linking with EDC greatly enhances detection of small RNA from both types of preparation¹⁴. Presumably, there will be a limit to the short RNA-binding capacity of nylon membrane and this might be approached when using highly enriched fractions. It has been suggested that UV cross-linking exploits free amine groups on nylon membrane⁵. Therefore, EDC cross-linking is likely to have a similar upper binding limit as UV cross-linking.

Choice of buffer. Tris has primary amine groups and, to avoid any potential reaction with EDC, we use MOPS–NaOH (pH 7) to buffer our dPAGs instead of conventional Tris–Borate–EDTA (TBE). MOPS–NaOH (pH 7)-buffered gels appear to be slightly less denaturing than TBE gels and often we find the longer pre-miRNA molecules (70 nt) run faster than expected at 50 nt. To get accurate sizing of the larger molecules, the MOPS–NaOH (pH 7)-buffered gels can be run at higher temperatures to aid denaturation. We have observed that pre-miRNA run at their predicted size when MOPS–NaOH (pH 7) gels are run hot with buffer preheated (50–55 °C) at 400 V (run time is reduced to 2 h). We have not thoroughly compared the efficiency of EDC cross-linking post-MOPS–NaOH (pH 7) or TBE-buffered dPAGE directly. Others have separated RNA using TBE-buffered dPAGE and then satisfactorily used our EDC cross-linking protocol¹⁸, but no comparison with using a MOPS system was described.

Use of radioactive size markers. Running [³²P] ATP-labeled Decade marker RNA is of obvious importance for the correct estimation of the size of sample RNAs. They also provide a very useful 'yardstick' for monitoring the progress of the protocol:

• Imaging the marker before transfer (see Step 15) allows assessment of the quality of the gel electrophoresis.
• Hand-held Geiger monitoring of the membrane (see Step 21) allows simple monitoring of the progress of the blot and helps to optimize the transfer time.
• Imaging of the membrane after washing away EDC solution and comparing this with the image of the gel allows one to estimate the degree of RNA loss up to this point and whether it is disproportionate for particular sizes of RNA.
• Comparison of the marker image after hybridization and wash with that obtained immediately after cross-linking allows one to determine loss of RNA during the (usually) former steps.
• If one is reusing the blot for multiple probes (i.e., by sequential probing, imaging and stripping), one can monitor the useful life span of the blot by carefully comparing marker RNA images in serial images (also accounting for radioactive decay). This is useful as probe stripping usually employs high temperatures, which in our experience accelerates loss of RNA from a membrane.

Choice of membrane. We routinely use the neutral nylon membrane, Hybond NX (Amersham/Pharmacia). In principle, other nylon 6,6 neutral membranes from different manufactures should work as effectively, but we have not tested this. We also tested the positively charged nylon membrane, Hybond N+ (Amersham/Pharmacia), and found that EDC cross-linking afforded only slightly enhanced short RNA detection compared with UV.

Optimization of EDC cross-linking parameters for your small RNA of interest. The strategy described in Figure 2 legend can be used to optimize EDC cross-linking conditions to achieve the best detection levels for any small RNA of interest. In brief, identical amounts of the same RNA sample are loaded on a 10–15% dPAG. The same volume of [³²P] ATP-labeled Decade markers is loaded in each adjacent lane. After electrophoresis and transfer to one sheet of nylon membrane, strips containing one individual sample RNA lane and one adjacent marker RNA lane are cut and each cross-linked with EDC at a range of times and temperatures to be tested, for example, 60 °C for 15 min, 1 or 2 h (UV cross-linking can be included to see the overall benefit of using EDC cross-linking). All membranes are then hybridized in the same hybridization solution containing a probe complementary to the small RNA of interest. The improvement in detection, using EDC cross-linking, of the small RNA of interest can be gauged by comparing the posthybridization signal from each condition tested.

Figure 2: Enhanced detection of short RNA with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linking versus UV cross-linking.

Identical 20 g aliquots of total RNA from undifferentiated murine embryonic stem cells were loaded in multiple lanes of the same denaturing polyacrylamide gel (dPAG). The same volume of [³²P] ATP-labeled Decade markers were loaded in each adjacent lane. After electrophoresis and transfer to one sheet of nylon membrane, strips containing one individual sample RNA lane and one adjacent marker RNA lane were cut and each was cross-linked with 1,200 J UV (auto-cross-linking setting) or EDC at 60 °C for 15 min, 1 or 2 h. All membranes were then hybridized in the same hybridization solution containing a probe complementary to mmu-miR-292-3p. The improvement of detection of the microRNA after EDC cross-linking for 2 h was 30-fold. Blue arrow, bromophenol blue dye front from 6 loading dye. Green arrow, xylene cyanol dye in Decade marker loading dye. Black arrow, mmu-miR-292-3p. M, [³²P]ATP-labeled Decade marker. S, RNA sample.

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DNA oligo dot blot control. In small RNA northern blot hybridizations, we routinely include a separate membrane onto which we have pipetted dilutions of DNA oligonucleotides that are complementary to our probe ('DNA oligo dot blot': see TROUBLESHOOTING). These are simple to prepare but are very useful positive controls that should give a strong positive signal if the probe has been synthesized correctly and if hybridization conditions were sufficiently tolerant. As DNA oligos are manufactured least expensively without terminal phosphates, we use UV cross-linking to fix these to nylon membranes. If the 'DNA oligo dot blot' control is positive and the northern blot has weak or no signal, one can then infer that something has gone wrong with the production of the small RNA northern blot (see TROUBLESHOOTING).

Materials

Reagents

• TRI-reagent (Sigma, cat. no. T9424)
  Caution Contains phenol and guanidinium thiocyanate. Wear lab coat, gloves and safety spectacles when handling.
• Diethylpyrocarbonate (DEPC; Sigma, cat. no. D5758)
• Deionized formamide (Sigma, cat. no. F9037)
• Acrylamide/bis (19:1) (Sigma, cat. no. A2917)
  Caution Neurotoxic. Wear lab coat, gloves and safety spectacles when handling.
• Urea (Fluka, cat. no. 51461)
• Tetramethylethylenediamine(Sigma, cat. no. T9281)
• Ammonium persulfate (Sigma, cat. no. A3678)
• MOPS (Roche, cat. no. 11 124 684 001)
• Ethidium bromide (EtBr; Sigma, cat. no. E1510) (see REAGENT SETUP)
  Caution Intercalating agent. Carcinogenic and mutagenic. Wear lab coat, gloves and safety spectacles when handling.
• Nylon membrane (Hybond NX; Amersham/Pharmacia, cat. no. RPN303T)
• 3MM Whatman chromatography paper (Schleicher & Schuell, cat. no. 3030 917)
• Decade RNA markers (Ambion, cat. no. 7778)
• [³²P] ATP (Amersham/Pharmacia, cat. no. AA0018)
  Caution Wear lab coat, gloves and safety spectacles when handling.
• 1-Methylimidazole (Sigma-Aldrich, cat. no. M50834)
• 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC: Sigma, cat. no. 7750) (see REAGENT SETUP)
  Critical All reagents listed here are the ones we use, but these or equivalent reagents are available from other manufacturers.

Equipment

• Vertical gel system (protean II Bio-Rad system with gel plates 16 20 cm; Bio-Rad)
  Critical We use equipment designed principally for protein analysis. Similar equipment is available from a number of different manufacturers.
• Semi-dry electroblotter (SciPlas) (see EQUIPMENT SETUP)
  Critical We have previously used a completely 'wet' electrotransfer unit satisfactorily, but this was slightly more difficult to set up properly and required several liters of transfer buffer.

Reagent setup

• DEPC-treated water

  • Add 1 ml DEPC to 1,000 ml distilled water. Shake vigorously to mix. Incubate, in fume hood, at room temperature (20 °C) overnight. Autoclave to inactivate residual DEPC and cool before use. DEPC-treated water can be stored at room temperature. Providing the DEPC-treated water remains contamination free, it can be stored and used until finished.

• 50 MOPS–NaOH (pH 7)

  • 1 M MOPS prepared in distilled water (pH 7) with NaOH. Store at 4 °C. This should be discarded if microbial and/or ribonuclease contamination is suspected or once the buffer acquires a noticeable yellow color.

• dPAG

  • Denaturing polyacrylamide (19:1) gel prepared with 10–15% acrylamide, 7 M urea and buffered with 20 mM MOPS–NaOH (pH 7) (see Table 1). For detailed instructions on how to prepare and pour dPAGs, refer to Sambrook and Russell¹⁵.

    Table 1: dPAG recipes.

    

    
    Full table
    
    

• [³²P] ATP-labeled Decade RNA markers

  • Follow manufacturer's (Ambion) instructions for labeling.
    Caution Decade markers radiolabeled with [³²P] ATP work with appropriate shielding.

• 6 Loading dye

  • 0.25% (wt/vol) Bromophenol blue dissolved in 30% (vol/vol) glycerol. This can be stored at - 20 °C for several months. Providing the 6 loading dye remains contamination free, it can be stored and used until finished. Optional: we have found that including 1 MOPS buffer (the same as the electrophoresis running buffer) in the samples can sometimes improve the resolution of small RNA, such as miRNA.

• EtBr working solution 0.5 g ml^{- 1}

  • Prepare fresh, dilute stock (10 mg ml^{- 1}) with 20 mM MOPS–NaOH (pH 7) to use at 0.5 g ml^{- 1}.

• EDC cross-linking solution

  • 0.16 M EDC prepared in 0.13 M 1-methylimidazole at pH 8. Add 245 l of 12.5 M 1-methylimidazole to 9 ml DEPC-treated water. Adjust pH to 8.0 with 1 M HCl (usually requires 300 l). This can be prepared 1–2 h before use and kept at room temperature. Immediately before use, add 0.753 g EDC and make the volume up to 24 ml with DEPC-treated water. This gives a working solution of 0.16 M EDC in 0.13 M 1-methylimidazole at pH 8 and is sufficient to saturate 320 cm² (20 16 cm²) of 3 MM Whatman paper. We use the EDC cross-linking solution immediately and we have not tested how storage affects it.

Equipment setup

• Semi-dry electroblotter

  • Place on ice to avoid overheating during transfer.

Procedure

1. Points from here (point 1) up to and including point 1 are related to Extraction of RNAIsolate intact RNA using TRI-reagent (or other available reagents; see EXPERIMENTAL DESIGN), following manufacturer's instructions, and resuspend RNA in DEPC-treated water. Typically, we attempt to resuspend in volumes of DEPC-treated water that will give concentrations of 1–2 g l^{- 1} of RNA.
2. Points from here (point 2) up to and including point 4 are related to Assessment of quantity and quality of RNAMeasure RNA concentration by spectrophotometry at 260 nm (e.g., with Nanodrop spectrophotometer).
   Critical step Typically, we require between 10 and 20 g of total RNA to run per lane of the dPAG, but this can be adjusted depending on the abundance of the RNA of interest.Pause Point RNA resuspended in DEPC-treated water can be stored at - 80 °C. We have successfully used samples that have been in storage for >1 year in our laboratory.
3. Add deionized formamide to a final concentration of 50% to help maintain RNA integrity¹⁹.
   Critical step This dilutes your initial RNA sample concentration. We typically use 100% deionized formamide and thus a volume equal to the DEPC-treated water used to resuspend the RNA (Step 1) is required. As a result, the concentration of the sample (Step 2) is halved.Pause Point RNA resuspended in 50% deionized formamide can be stored at - 20 °C. We have successfully used samples that have been in storage for >2 years in our laboratory.
4. Check the quality of RNA by conventional agarose gel electrophoresis followed by EtBr staining. We typically run 0.5–1 g of sample RNA resuspended in 50% deionized formamide to assess quality. For detailed instructions of how to pour, run and stain agarose gels refer to Sambrook and Russell¹⁵. The clear observation of distinct, 18S and 28S, rRNA and tRNA bands is a good indicator that the RNA sample is of good quality and not degraded (Fig. 3a).

   Figure 3: Examples of RNA preparations suitable for northern blot analysis.

   

   (a) Total RNA assessed on a 1% agarose gel: Total RNA isolated from mouse embryonic stem (ES) cells resuspended in 50% deionized formamide ran on a 1% agarose gel buffered with 0.5 Tris–Borate–EDTA (TBE). Each lane (1 and 2) loaded with 1 g of total RNA. (b) Total RNA assessed on a 15% dPAG: Total RNA isolated from ES cells resuspended in 50% deionized formamide ran on a 15% dPAG gel buffered with 20 mM MOPS–NaOH pH 7. Each lane (1 and 2) is loaded with 10 g of total RNA.

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5. Points from here (point 5) up to and including point 6 are related to Prepare dPAGTiming: 2–2.5 hWe use the Bio-Rad protean II vertical gel system with 20 16 cm² plates and 1.5-mm spacers. Assemble the plates following manufacturer's instructions. Make sure the glass plates are clean before assembly. In our laboratory, it is usual practice to clean glass plates first with 70% ethanol followed by a rinse in distilled water before use.
6. Use (19:1) acrylamide/bis to prepare 10 or 15% denaturing gel mix (as described in REAGENT SETUP and Table 1) and pour gel. Insert well comb that will accommodate RNA sample to be loaded. This may vary depending on the concentration of the RNA sample. For detailed instructions on how to prepare and pour dPAGs, refer to Sambrook and Russell¹⁵.
   Critical step 60 ml of Mix is sufficient to prepare a gel 200 160 1.5 mm.Pause Point Allow at least 2 h for gels to set before use. Alternatively, gels can be wrapped in Saran and left overnight at room temperature to set.
7. Points from here (point 7) up to and including point 12 are related to Running dPAGTiming: 2–16 hRemove well comb and prerun gel in running buffer, 20 mM MOPS–NaOH (pH 7) (prepared as described in REAGENT SETUP) at desired voltage for at least 10 min.
   Critical step We prerun and run gels at the same voltage. Gels can be run at high voltage (300–400 V), in which case the run will take 2–4 h, depending on polyacrylamide concentration. Alternatively, gels can be run overnight (16 h) at 60–90 V.
8. During gel prerun, prepare RNA samples (typically, we run 10- 20 g of RNA already resuspended in 50% deionized formamide, see Steps 1–3) by adding 6 loading dye (prepared as described in REAGENT SETUP) to samples and heat denature at 95 °C for 1–5 min followed by snap cooling on ice.
   Critical step As well as inhibiting renaturation of RNA structures, snap cooling samples also makes sample loading easier.
9. We also run [³²P] ATP-labeled Decade RNA markers (as described in REAGENT SETUP) for sizing bands and as a control (see EXPERIMENTAL DESIGN and TROUBLESHOOTING). Follow manufacturer's instructions to prepare [³²P] ATP-labeled Decade markers.
10. Just before loading RNA samples (i.e., while they are on ice after denaturing—Step 8), flush wells thoroughly with running buffer [20 mM MOPS–NaOH (pH 7)] injected using a syringe and needle.
    Critical step This step is necessary because as soon as the well comb is removed from the gel and running buffer is overlaid, urea begins to leach from the gel and accumulates in a barely visible layer over the bottom of each well. In our experience, loading sample on top of this negatively affects the resolution of RNA during electrophoresis.
11. Load RNA samples, typically 10–20 g of RNA (see Step 8), and markers, as recommended by the manufacturer (see Step 9), onto the gel and run the gel at the desired voltage (see Step 7).
    Critical step On a 15% dPAG, the blue bromophenol loading dye front runs at the equivalent rate to a 10-nt long RNA. The dye front for each sample should run in a near perfect straight line indicating a uniform electric field across the gel. Disturbance of the dye front is a possible indicator that either the gel or the run has not been uniform.
12. Disassemble the gel from the gel apparatus. Remove one glass plate from the gel, mark orientation of gel by cutting corner.
    Caution Running buffer is contaminated with [³²P] ATP-radiolabeled marker and urea—discard appropriately following safety guidelines.
13. Points from here (point 13) up to and including point 15 are related to Assessment of gel runTiming: 30 minAt this point, the gel is sitting on glass plate, so you can use this as support to handle gel or, with care, the gel can be lifted from the plate by hand. Place the gel in freshly prepared EtBr solution (prepared as described in REAGENT SETUP) for 5–10 min.
    Caution Do not use the electrophoresis running buffer from the gel run (Step 11) to prepare the EtBr staining solution. Although this might be economical and convenient, we have occasionally experienced problems with the subsequent electrophoretic transfer of the RNA from the gel to the nylon membrane that we have attributed to using 'recycled' running buffer at this stage.
    Critical step Flimsiness of gel increases with decreasing acrylamide concentration.
    Critical step Steps 13–15 are optional. If one is confident with dPAGE and the integrity of the sample, then one can proceed directly from Step 12 to 16.
14. Remove gel from EtBr solution and then scan stained gel to assess the run. We use a Fuji FLA5000 scanner, but a UV light box can also be used. A number of discrete, strongly staining bands >60 nt should be visible. These include tRNA (70–110 nt) and 5S RNA (120 nt). These stained RNA can be used to assess the relative loading of RNA samples retrospectively if a quantitative digital image is obtained. The clarity and sharpness of the tRNA bands is a good indicator of RNA quality and integrity during the electrophoresis (Fig. 3b).
    Critical step If the integrity of the tRNA bands is diminished (may stain weakly with a heavily stained smear down the lane), then one should suspect the quality of the RNA sample and recognize that data acquired following the protocol to completion may not be reliable.
15. Thereafter, wrap the gel in Saran and place, face up, with imaging screen. Expose to detect signal from the [³²P] ATP-labeled Decade marker; typically, an exposure of 3–5 min is sufficient. Separation of the marker into its respective fragments is a good indicator that gel electrophoresis has also successfully separated the RNA samples (Fig. 4). This is useful if EtBr staining of RNA samples is weak.

    Figure 4: Use of [³²P] ATP-labeled Decade markers as a control for small RNA northern blotting procedure.

    

    Signal from [³²P] ATP-labeled Decade markers ran on 15% dPAG gel buffered with 20 mM MOPS–NaOH pH 7 and then transferred onto nylon membrane followed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linking: (a) gel image, (b) gel image after transfer, (c) nylon membrane image after transfer and (d) nylon membrane image after EDC cross-linking.

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16. Points from here (point 16) up to and including point 23 are related to Transfer of RNA from dPAG onto nylon membraneTiming: 1 hPrewet nylon membrane (200 160 mm) with distilled water and place it on the gel. Layer three sheets (200 160 mm) of 3MM Whatman (prewet with distilled water) on top of the nylon membrane. With each layer added, roll out any bubbles between the gel, membrane and Whatman.
17. Lift the gel, nylon membrane and Whatman, and place on the positive electrode of the semi-dry electroblotter with gel uppermost. Add another three sheets of 3MM Whatman (prewet with distilled water) on top of the gel; with each layer added, roll out bubbles.
    Critical step The exact composition of the blot (e.g., the number of layers of 3MM) may be varied according to user's preference; however, the key issue is to ensure that the membrane is tightly and uniformly apposed to the gel and is between the gel and the positive electrode.
18. Place negative electrode lid on to transfer sandwich: 3MM–gel–membrane–3MM (see Fig. 1, and Supplementary Fig. 1, online).
19. Place transfer unit on ice and transfer RNA by applying 20 V for 30–60 min.
    Critical step Little or no [³²P] ATP-labeled Decade marker RNA (i.e., radioactivity) should pass through the membrane to the 3MM or electrode. It is possible to 'over-transfer' by blotting for too long. This will be indicated by significant amounts of radioactive marker RNA being deposited on the 3MM below the membrane and/or the positive electrode of the transfer unit. In our experience, over-transfer also leads to poor signal from the sample RNA. Simply peeling back the gel partially from the membrane and monitoring each allows one to check the progress of the gel nondisruptively. The gel is gently rolled back into place after this check and transfer resumed.
20. After transfer is complete, remove layers of 3MM Whatman sitting on top of gel without disturbing the gel and membrane. Mark the position of the wells on the nylon membrane using pencil (does not run or wash off in subsequent hybridization steps).
21. Remove the gel and keep it for post-transfer exposure (see Step 23). The majority of the bromophenol blue should be on the membrane, an indication that transfer has occurred. Additionally, the gel and membrane can be monitored using a Geiger counter; the majority of counts should be on the membrane.
22. Carefully lift the membrane and avoid all contact with the side onto which the RNA has been deposited.
23. Optional: Wrap both the gel and membrane in Saran and expose both to an imaging screen for the same time as in Step 15. Upon successful transfer, the majority of signal from the [³²P] ATP-labeled Decade marker should now be on the membrane (compare Fig. 4b with c).
    Critical step In our experience, wrapping the membrane in Saran wrap does not disturb the position of the transferred RNA.
24. Points from here (point 24) up to and including point 27 are related to EDC cross-linkingTiming: 15 min to 2 hPlace the damp membrane with RNA side face up onto 3MM (210 170 mm) saturated in freshly prepared cross-linking EDC reagent (prepared as described in REAGENT SETUP).
25. Wrap membrane and 3MM in Saran and incubate at 50–60 °C for up to 2 h. We have found that the optimum cross-linking time can vary for different short RNA and so can be optimized by the user (see Fig. 2). A good starting point is 1 h at 60 °C.
    Critical step As a precaution, the RNA side should not be in direct contact with the saturated 3MM and excess cross-linking reagent should not be washed over the RNA face of the membrane.
26. After cross-linking, rinse membrane in excess RNase-free distilled water to remove any residual cross-linking solution.
27. Optional: To check for any loss of RNA during EDC cross-linking (Steps 24–26), expose cross-linked northern blot from Step 27 to imaging screen (as described in Step 15) and compare Decade marker signal to post-transfer signal (Step 23) (compare Fig. 4c with d).Pause Point After the membrane has been rinsed to remove the EDC solution, it can be air-dried and stored wrapped in Saran at - 20 °C until required for hybridization.Troubleshooting

Timing

Steps 1–4, RNA extraction, quantification, assessment of integrity: depends on the number of samples, can be stopped and resumed
Steps 5 and 6, preparation of dPAG: 20 min, allow to set for a minimal 2 h or can be poured on the day before use
Steps 7–15, prerun and running gel: 2 h or overnight 16 h
Steps 16–23, transfer: 1 h
Steps 24–27, EDC cross-linking: up to 2 h
Steps 7–27 should be done sequentially

Troubleshooting

Troubleshooting is generally well established for northern blotting¹⁵. Table 2 lists problems that might particularly arise from the use of EDC rather than UV cross-linking after a successful separation of RNA samples using dPAGE. We also briefly mention problems that can be mistaken for a failure in EDC cross-linking, but which may be due to steps that follow successful cross-linking, for example, probe synthesis and hybridization. Comprehensive protocols and troubleshooting guides for these particular steps are available elsewhere. As part of our controls, we run [³²P] ATP-labeled Decade RNA markers; these allow us to assess each step of the protocol before and after cross-linking (Fig. 4, also see EXPERIMENTAL DESIGN and Table 2). To evaluate the benefit of EDC cross-linking for novel small RNA, we run samples in duplicate and cross-link them with EDC or UV for comparison.

Table 2: Troubleshooting table.

Full table

Anticipated results

The enhanced sensitivity achieved using EDC cross-linking will improve the detection of small RNA and thus correlation of specific RNA with biological processes. Figures 2, 3, 4 demonstrate the expected results from each step of the small northern blot protocol. Success of each step is important for the final enhanced detection of small RNA with EDC cross-linking. Figure 3 demonstrates resolution of total RNA after (a) agarose gel electrophoresis and (b) dPAGE. Figure 4 demonstrates the signal from [³²P] ATP-labeled Decade marker in the dPAG after (a) electrophoresis, (b) gel and (c) nylon membrane after transfer and (d) nylon membrane after cross-linking. Figure 2 demonstrates the enhanced detection of short RNA (mmu-miR-292-3p) with EDC cross-linking versus UV cross-linking.

Note: Supplementary information is available via the HTML version of this article.