- Consists of 9 RNA fragments ranging in the size from 0.5-10 kb
- 1.5 kb reference band is ~2-fold brighter for easy orientation and band size determination
- Designed for use with formaldehyde or glyoxal agarose gels
- Suitable for use as a control template for cDNA synthesis as all RNA fragments in the ladder contain a 40 nucleotide poly A tail
- Visualized with ethidium bromide (EtBr) or SYBR® Green II staining
Storage Buffer: 10 mM HEPES, pH 7.2; 2 mM EDTA
Storage: -20°C (for 2 months); -80°C (long-term)
Stability: 6 months at -80°C
Guidelines for Preventing RNase Contamination
- To avoid repeated freezing and thawing, aliquot the ladder in small volumes and store at -80°C
- Always wear gloves, use sterile, RNase-free tips, tubes and plasticware, and use aseptic techniques
Thaw the 0.5-10 Kb RNA Ladder on ice and mix gently to ensure the ladder is homogenous. Avoid vortexing the ladder. To avoid staining/destaining steps, you may add RNase-free EtBr solution (cat. no. 15585-011) to a final concentration of 10-50 μg/ml to the ladder.
Formaldehyde Denaturing Gels
- Pre-run a horizontal 1.2-1.5% agarose gel (2-3 mm, lane width) containing 6% (w/v) formaldehyde, 20 mM MOPS (pH 7.0), 1 mM EDTA submerged in buffer (20 mM MOPS, pH 7.0; 1 mM EDTA) for 10 minutes at 100 V.
- To 3 μg (3 μl) of ladder, add loading buffer (recommended final loading buffer concentration is 20 mM MOPS; pH 7.0, 6% w/v formaldehyde, 31% v/v deionized formamide, 1 mM EDTA, 0.0125% xylene cyanol and 0.0125% bromophenol blue) and mix.
- Denature the ladder in loading buffer at 70°C for 10 minutes. Keep on ice for 1-2 minutes to quench.
- Load ladder and your RNA samples on the pre-run gel from Step 1.
- Perform electrophoresis at 100 V until the bromophenol blue dye has migrated two-thirds the gel length.
- Stain the RNA ladder with:
- 0.5 μg/ml EtBr solution for 10 minutes and detain the gel in deionized water for 10 minutes OR
- 1:10000 dilution of SYBR® Green II RNA Gel Stain (cat. no. S7564) in TBE for 20-40 minutes. No destaining is required.
- Visualize ladder bands on a UV transilluminator (302 nm) and photograph using a gel documentation system. The RNA bands may fade and disappear on prolonged exposure to UV light.
Native TBE/TAE Agarose Gels
For 1.5% native TBE or TAE agarose gels, use 0.8 μg (8 μl of 1:10 dilution RNA ladder per lane (2-3 mm lane width). Denature the ladder, perform electrophoresis, and visualize as described above.
- Denature 3 μg (3 μl) RNA Ladder in loading buffer (recommended final loading buffer concentration is 1 M deionized glyoxal, 50% (v/v) DMSO, 10 mM sodium phosphate (pH 7.0), 0.01% xylene cyanol, 0.01% bromophenol blue for 30 minutes at 50°C. Keep on ice for 1-2 minutes to quench.
- Load your samples on a horizontal 1.5% agarose gel containing 10 mM sodium phosphate (pH 7.0), 2 mg/ml sodium iodoacetate. Run the gel submerged in 10 mM sodium phosphate (pH 7.0) at 160 V for 2 h.
- Stain with EtBr or SYBR® Green II, visualize, and image the gel.
- Incubate 2 μg (2 μl) RNA ladder with 5 μg anchored Oligo(dT)20 primer at 70°C for 5 minutes. Keep on ice.
- Combine the RNA-oligo dT complex from Step 1 with 1X 1st strand cDNA synthesis buffer supplemented with 10 mM DTT, 500 μM dNTP, 2 U RNaseOUT™ (cat. no.10777-019), 1-3 μCi α-P32-dCTP (3000Ci/mmole) or Chromatide®Alexa Fluor® 546-14-UTP (cat. no. C-11404) in a reaction volume of 18 μl.
- Add 400 U of SuperScript™ III Reverse Transcriptase (cat. no.18080-093). Adjust the final reaction volume to 20 μl. Incubate at 50°C for 1 hour.
- Add 10 μl 0.5 M EDTA, pH 8.0 to terminate the reaction and then add 20 μl STE buffer (150 mM NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA).
- Remove unincorporated NTPs by gel filtration with G-50 columns.
- Mix a fraction of the purified cDNA reaction with 10X alkaline agarose loading dye (300 mM NaOH, 2 mM EDTA, 20% glycerol, 10% saturated thymol blue).
- Analyze samples on a 1.2% alkaline agarose gel containing 30 mM NaOH, 2 mM EDTA, pH 7.5 and electrophoresed using 30 mM NaOH, 2 mM EDTA, pH 7.5.
- Dry the gel in a heated vacuum drier for 1.5 hours and expose the gel to a film or a phosphoimager plate
Formaldehyde gel analysis must clearly show 9 bands between 0.5-10 kb and the 1.5 kb band must be more intense than other ladder bands.